Method and apparatus for determining a probability of colorectal cancer in a subject

ABSTRACT

A method of determining a probability that a human test subject has colorectal cancer as opposed to not having colorectal cancer is disclosed. The method comprises, for each gene of a set of one or more genes selected from the group consisting of ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1: determining a level of RNA encoded by the gene in blood of the test subject, thereby generating test data; providing positive control data representing levels of RNA encoded by the gene in blood of human control subjects having colorectal cancer, and providing negative control data representing levels of RNA encoded by the gene in blood of human control subjects not having colorectal cancer; and determining a probability that the test data corresponds to the positive control data and not to the negative control data, where the probability that the test data corresponds to the positive control data and not to the negative control data represents the probability that the test subject has colorectal cancer as opposed to not having colorectal cancer.

This application is a continuation application of U.S. non-provisionalapplication Ser. No. 12/384,914, filed Apr. 10, 2009, which itselfclaims the benefit of U.S. provisional application 61/123,798, filedApr. 10, 2008 and of U.S. provisional application 61/123,831, filed Apr.11, 2008. The entire contents of the non-provisional application andboth provisional applications are incorporated by reference herein.

TECHNICAL FIELD

The disclosure relates to apparatuses, kits and methods for determininga probability of colorectal cancer in a test subject. More particularly,the disclosure relates to apparatuses, kits and methods for diagnosingcolorectal cancer in a test subject by measuring a level of one or moregene products in blood of the test subject.

BACKGROUND

Colorectal cancer causes 655,000 deaths worldwide per year, making itthe second-leading cause of cancer-related deaths. It is the third mostfrequently diagnosed cancer in men and women in the United States andcarries an overall population lifetime risk of 6%. (American CancerSociety. Cancer Facts and Figures. 2008. Atlanta: American CancerSociety). The American Cancer Society estimates that about 108,070 newcases of colon cancer (53,760 in men and 54,310 in women) and 40,740 newcases of rectal cancer (23,490 in men and 17,250 in women) will bediagnosed in 2008. Of those diagnosed, nearly half are expected to diewithin five years. In the United States in 2008 an estimated 50,000 menand women will die of cancer of the colon and rectum. (American CancerSociety 2008). This high mortality rate is due at least in part to thefact that a large proportion of cancers are detected at relatively latestages, such as following onset of overt symptoms, when the cancer ismore difficult to treat. In addition, identification of colorectalcancer at later stages concomitantly necessitates harsher treatment,such as radical colostomy. It has been shown that the identification andtreatment of colorectal cancer at earlier stages significantly reducesthe risk of developing more advanced disease, and hence risk of deathfrom the disease. Stage at detection is critically related to patientsurvival. Localized cancers (Dukes's Stage A or B) have an excellentprognosis of 82%-93% at five years. Regional (Dukes's Stage C) patientshave a five year survival rates of 55% to 60%; and only 5% to 8% ofpatients with late stage cancer will survive the five year span.(O'Connell J B, Maggard M, Ko C Y. Colon cancer survival rates with thenew American Joint Committee on cancer sixth edition staging. JNCI.2004; 96: 1420-1425). Therefore, a test to screen for colorectal cancerso as to allow earlier treatment should markedly reduce the incidence ofadvanced-stage colorectal cancer (Ransohoff D F. Colorectal cancerscreening in 2005: status and challenges. Gastroenterology. 2005 May;128(6):1685-95) and decrease the current costs to the medical system.Thus, the American Cancer Society recommends that all Americans age 50and older be screened regularly for colorectal cancer. Unfortunately,only a small fraction of the population at risk is screened for thedisease (Mitka M. Colorectal cancer screening rates still fall far shortof recommended levels. JAMA. 2008 Feb. 13; 299(6):622), as currentlyavailable screening methods require insufficiently available and/orcostly resources, are associated with unacceptably low patientcompliance, and/or are associated with significant health risks.

Currently utilized screening technologies to test for colorectal cancerinclude fecal occult blood test (FOBT), flexible sigmoidoscopy, doublecontrast barium enema (DCBE), and colonoscopy. The current recommendedstandards for screening for colorectal cancer in individuals over theage of 50 and who are considered part of an average risk populationinclude: an FOBT yearly, a sigmoidoscopy every five years, a colonoscopyevery ten years and a DCBE every five years. For a high risk populationwhere one or more family members have had colorectal cancer, acolonoscopy is recommended every two years as a follow up to FOBT orsigmoidoscopy. Each of these tests suffers significant disadvantages.Fecal occult blood testing suffers from low sensitivity, requiressignificant dietary and other restrictions prior to testing and isassociated with poor patient compliance. Sigmoidoscopy and colonoscopyare more sensitive than the other standard methods since they involvedirect visualization of the lumen of the colon, however these methodsare also associated with various significant disadvantages.Sigmoidoscopy and colonoscopy are both highly invasive procedures whichcause significant levels of discomfort, causing many individuals to optnot to undergo these recommended screening procedures. Sigmoidoscopyonly allows visualization of the distal part of the colon and hencecannot detect a relatively large fraction of cancers, and colonoscopy,despite allowing examination essentially along the entire length of thecolon, is associated with a significant failure rate for detection ofcolorectal cancer. In addition, sigmoidoscopy and colonoscopy arecostly, are insufficiently available, and may result in potentiallylethal complications, such as accidental intestinal perforation.

Various approaches have been proposed in the prior art for colorectalcancer testing using identification and analysis of markers of thisdisease in blood (reviewed in Hundt S. et al. Blood markers for earlydetection of colorectal cancer: a systematic review. Cancer EpidemiolBiomarkers Prey. 2007 October; 16(10):1935-53). Such approaches, ifsuccessful, would have the advantage of circumventing criticaldisadvantages of the standard prior art methods, by virtue, for example,of being relatively non-invasive, minimally cumbersome, essentiallyrisk-free and hence likely to be associated with increased patientscreening compliance rates. However, none of these approaches hasdemonstrated an optimal capacity for diagnosing colorectal cancer.

Thus, there is a longstanding and urgent need for an improved method ofdetermining a probability of colorectal cancer in a subject based onanalysis of blood markers.

SUMMARY

The invention discloses novel methods, apparatuses and kits fordetermining a probability of colorectal cancer in a subject, based onnovel blood markers of colorectal cancer. This use can be effected in avariety of ways as further described and exemplified herein.

According to one aspect of the invention there is provided a method ofdetermining a probability that a human test subject has colorectalcancer as opposed to not having colorectal cancer, the methodcomprising, for each gene of a set of one or more genes selected fromthe group consisting of ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 andVNN1: (a) determining a level of RNA encoded by the gene in blood of thetest subject, thereby generating test data; (b) providing positivecontrol data representing levels of RNA encoded by the gene in blood ofhuman control subjects having colorectal cancer, and providing negativecontrol data representing levels of RNA encoded by the gene in blood ofhuman control subjects not having colorectal cancer; and (c) determininga probability that the test data corresponds to the positive controldata and not to the negative control data, wherein the probability thatthe test data corresponds to the positive control data and not to thenegative control data represents the probability that the test subjecthas colorectal cancer as opposed to not having colorectal cancer.

According to another aspect of the invention there is provided a methodof determining a probability that a human test subject has colorectalcancer as opposed to not having colorectal cancer, the methodcomprising: (a) determining a level of RNA encoded by a ANXA3 gene inblood of the test subject, thereby generating test data; (b) providingpositive control data representing levels of RNA encoded by the gene inblood of human control subjects having colorectal cancer, and providingnegative control data representing levels of RNA encoded by the gene inblood of human control subjects not having colorectal cancer; and (c)determining a probability that the test data corresponds to the positivecontrol data and not to the negative control data, wherein theprobability that the test data corresponds to the positive control dataand not to the negative control data represents the probability that thetest subject has colorectal cancer as opposed to not having colorectalcancer.

According to yet another aspect of the invention there is provided amethod of determining a probability that a human test subject hascolorectal cancer as opposed to not having colorectal cancer, the methodcomprising: (a) determining a level of RNA encoded by a CLEC4D gene inblood of the test subject, thereby generating test data; (b) providingpositive control data representing levels of RNA encoded by the gene inblood of human control subjects having colorectal cancer, and providingnegative control data representing levels of RNA encoded by the gene inblood of human control subjects not having colorectal cancer; and (c)determining a probability that the test data corresponds to the positivecontrol data and not to the negative control data, wherein theprobability that the test data corresponds to the positive control dataand not to the negative control data represents the probability that thetest subject has colorectal cancer as opposed to not having colorectalcancer.

According to one aspect of the invention there is provided a method ofdetermining a probability that a human test subject has colorectalcancer as opposed to not having colorectal cancer, the methodcomprising: (a) determining a level of RNA encoded by a IL2RB gene inblood of the test subject, thereby generating test data; (b) providingpositive control data representing levels of RNA encoded by the gene inblood of human control subjects having colorectal cancer, and providingnegative control data representing levels of RNA encoded by the gene inblood of human control subjects not having colorectal cancer; and (c)determining a probability that the test data corresponds to the positivecontrol data and not to the negative control data, wherein theprobability that the test data corresponds to the positive control dataand not to the negative control data represents the probability that thetest subject has colorectal cancer as opposed to not having colorectalcancer.

According to still another aspect of the invention there is provided amethod of determining a probability that a human test subject hascolorectal cancer as opposed to not having colorectal cancer, the methodcomprising: (a) determining a level of RNA encoded by a LMNB1 gene inblood of the test subject, thereby generating test data; (b) providingpositive control data representing levels of RNA encoded by the gene inblood of human control subjects having colorectal cancer, and providingnegative control data representing levels of RNA encoded by the gene inblood of human control subjects not having colorectal cancer; and (c)determining a probability that the test data corresponds to the positivecontrol data and not to the negative control data, wherein theprobability that the test data corresponds to the positive control dataand not to the negative control data represents the probability that thetest subject has colorectal cancer as opposed to not having colorectalcancer.

According to a further aspect of the invention there is provided amethod of determining a probability that a human test subject hascolorectal cancer as opposed to not having colorectal cancer, the methodcomprising: (a) determining a level of RNA encoded by a PRRG4 gene inblood of the test subject, thereby generating test data; (b) providingpositive control data representing levels of RNA encoded by the gene inblood of human control subjects having colorectal cancer, and providingnegative control data representing levels of RNA encoded by the gene inblood of human control subjects not having colorectal cancer; and (c)determining a probability that the test data corresponds to the positivecontrol data and not to the negative control data, wherein theprobability that the test data corresponds to the positive control dataand not to the negative control data represents the probability that thetest subject has colorectal cancer as opposed to not having colorectalcancer.

According to yet a further aspect of the invention there is provided amethod of determining a probability that a human test subject hascolorectal cancer as opposed to not having colorectal cancer, the methodcomprising: (a) determining a level of RNA encoded by a TNFAIP6 gene inblood of the test subject, thereby generating test data; (b) providingpositive control data representing levels of RNA encoded by the gene inblood of human control subjects having colorectal cancer, and providingnegative control data representing levels of RNA encoded by the gene inblood of human control subjects not having colorectal cancer; and (c)determining a probability that the test data corresponds to the positivecontrol data and not to the negative control data, wherein theprobability that the test data corresponds to the positive control dataand not to the negative control data represents the probability that thetest subject has colorectal cancer as opposed to not having colorectalcancer.

According to still a further aspect of the invention there is provided amethod of determining a probability that a human test subject hascolorectal cancer as opposed to not having colorectal cancer, the methodcomprising: (a) determining a level of RNA encoded by a VNN1 gene inblood of the test subject, thereby generating test data; (b) providingpositive control data representing levels of RNA encoded by the gene inblood of human control subjects having colorectal cancer, and providingnegative control data representing levels of RNA encoded by the gene inblood of human control subjects not having colorectal cancer; and (c)determining a probability that the test data corresponds to the positivecontrol data and not to the negative control data, wherein theprobability that the test data corresponds to the positive control dataand not to the negative control data represents the probability that thetest subject has colorectal cancer as opposed to not having colorectalcancer.

According to further features of the invention described below, thedetermining of the level of RNA encoded by the gene in blood of the testsubject is effected by determining the level of RNA encoded by the genein a blood sample isolated from the test subject.

According to further features of the invention described below, themethod of determining the probability that the human test subject hascolorectal cancer as opposed to not having colorectal cancer furthercomprises determining levels of RNA encoded by the gene in blood of apopulation of human subjects having colorectal cancer, thereby providingthe positive control data representing the levels of RNA encoded by thegene in blood of human control subjects having colorectal cancer, anddetermining levels of RNA encoded by the gene in blood of a populationof human subjects not having colorectal cancer, thereby providing thenegative control data representing the levels of RNA encoded by the genein blood of human control subjects not having colorectal cancer.

According to further features of the invention described below, thedetermining of the probability that the test data corresponds to thepositive control data and not to the negative control data is effectedby applying to the test data a mathematical model derived from thepositive control data and from the negative control data, wherein themathematical model is for determining the probability that datarepresenting a level of RNA encoded by the gene corresponds to thepositive control data and not to the negative control data.

According to another aspect of the invention there is provided acomputer-based method of determining a probability that a human testsubject has colorectal cancer as opposed to not having colorectalcancer, from test data representing a level of RNA encoded by a ANXA3gene in blood of the test subject, the method comprising, for each geneof a set of one or more genes selected from the group consisting ofANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1computer-implemented steps of: (a) applying to the test data amathematical model derived from positive control data representinglevels of RNA encoded by the gene in blood of human control subjectshaving colorectal cancer, and from negative control data representinglevels of RNA encoded by the gene in blood of human control subjects nothaving colorectal cancer, wherein the mathematical model is fordetermining a probability that data representing a level of RNA encodedby the gene corresponds to the positive control data and not to thenegative control data; and (b) outputting the probability that datarepresenting a level of RNA encoded by the gene corresponds to thepositive control data and not to the negative control data, wherein theprobability that the test data corresponds to the positive control dataand not to the negative control data represents the probability that thetest subject has colorectal cancer as opposed to not having colorectalcancer.

According to another aspect of the invention there is provided acomputer-based method of determining a probability that a human testsubject has colorectal cancer as opposed to not having colorectalcancer, from test data representing a level of RNA encoded by a ANXA3gene in blood of the test subject, the method comprisingcomputer-implemented steps of: (a) applying to the test data amathematical model derived from positive control data representinglevels of RNA encoded by the gene in blood of human control subjectshaving colorectal cancer, and from negative control data representinglevels of RNA encoded by the gene in blood of human control subjects nothaving colorectal cancer, wherein the mathematical model is fordetermining a probability that data representing a level of RNA encodedby the gene corresponds to the positive control data and not to thenegative control data; and (b) outputting the probability that datarepresenting a level of RNA encoded by the gene corresponds to thepositive control data and not to the negative control data, wherein theprobability that the test data corresponds to the positive control dataand not to the negative control data represents the probability that thetest subject has colorectal cancer as opposed to not having colorectalcancer.

According to another aspect of the invention there is provided acomputer-based method of determining a probability that a human testsubject has colorectal cancer as opposed to not having colorectalcancer, from test data representing a level of RNA encoded by a ANXA3gene in blood of the test subject, the method comprisingcomputer-implemented steps of: (a) applying to the test data amathematical model derived from positive control data representinglevels of RNA encoded by the gene in blood of human control subjectshaving colorectal cancer, and from negative control data representinglevels of RNA encoded by the gene in blood of human control subjects nothaving colorectal cancer, wherein the mathematical model is fordetermining a probability that data representing a level of RNA encodedby the gene corresponds to the positive control data and not to thenegative control data; and (b) outputting the probability that datarepresenting a level of RNA encoded by the gene corresponds to thepositive control data and not to the negative control data, wherein theprobability that the test data corresponds to the positive control dataand not to the negative control data represents the probability that thetest subject has colorectal cancer as opposed to not having colorectalcancer.

According to another aspect of the invention there is provided acomputer-based method of determining a probability that a human testsubject has colorectal cancer as opposed to not having colorectalcancer, from test data representing a level of RNA encoded by a ANXA3gene in blood of the test subject, the method comprisingcomputer-implemented steps of: (a) applying to the test data amathematical model derived from positive control data representinglevels of RNA encoded by the gene in blood of human control subjectshaving colorectal cancer, and from negative control data representinglevels of RNA encoded by the gene in blood of human control subjects nothaving colorectal cancer, wherein the mathematical model is fordetermining a probability that data representing a level of RNA encodedby the gene corresponds to the positive control data and not to thenegative control data; and (b) outputting the probability that datarepresenting a level of RNA encoded by the gene corresponds to thepositive control data and not to the negative control data, wherein theprobability that the test data corresponds to the positive control dataand not to the negative control data represents the probability that thetest subject has colorectal cancer as opposed to not having colorectalcancer.

According to another aspect of the invention there is provided acomputer-based method of determining a probability that a human testsubject has colorectal cancer as opposed to not having colorectalcancer, from test data representing a level of RNA encoded by a ANXA3gene in blood of the test subject, the method comprisingcomputer-implemented steps of: (a) applying to the test data amathematical model derived from positive control data representinglevels of RNA encoded by the gene in blood of human control subjectshaving colorectal cancer, and from negative control data representinglevels of RNA encoded by the gene in blood of human control subjects nothaving colorectal cancer, wherein the mathematical model is fordetermining a probability that data representing a level of RNA encodedby the gene corresponds to the positive control data and not to thenegative control data; and (b) outputting the probability that datarepresenting a level of RNA encoded by the gene corresponds to thepositive control data and not to the negative control data, wherein theprobability that the test data corresponds to the positive control dataand not to the negative control data represents the probability that thetest subject has colorectal cancer as opposed to not having colorectalcancer.

According to another aspect of the invention there is provided acomputer-based method of determining a probability that a human testsubject has colorectal cancer as opposed to not having colorectalcancer, from test data representing a level of RNA encoded by a ANXA3gene in blood of the test subject, the method comprisingcomputer-implemented steps of: (a) applying to the test data amathematical model derived from positive control data representinglevels of RNA encoded by the gene in blood of human control subjectshaving colorectal cancer, and from negative control data representinglevels of RNA encoded by the gene in blood of human control subjects nothaving colorectal cancer, wherein the mathematical model is fordetermining a probability that data representing a level of RNA encodedby the gene corresponds to the positive control data and not to thenegative control data; and (b) outputting the probability that datarepresenting a level of RNA encoded by the gene corresponds to thepositive control data and not to the negative control data, wherein theprobability that the test data corresponds to the positive control dataand not to the negative control data represents the probability that thetest subject has colorectal cancer as opposed to not having colorectalcancer.

According to another aspect of the invention there is provided acomputer-based method of determining a probability that a human testsubject has colorectal cancer as opposed to not having colorectalcancer, from test data representing a level of RNA encoded by a ANXA3gene in blood of the test subject, the method comprisingcomputer-implemented steps of: (a) applying to the test data amathematical model derived from positive control data representinglevels of RNA encoded by the gene in blood of human control subjectshaving colorectal cancer, and from negative control data representinglevels of RNA encoded by the gene in blood of human control subjects nothaving colorectal cancer, wherein the mathematical model is fordetermining a probability that data representing a level of RNA encodedby the gene corresponds to the positive control data and not to thenegative control data; and (b) outputting the probability that datarepresenting a level of RNA encoded by the gene corresponds to thepositive control data and not to the negative control data, wherein theprobability that the test data corresponds to the positive control dataand not to the negative control data represents the probability that thetest subject has colorectal cancer as opposed to not having colorectalcancer.

According to another aspect of the invention there is provided acomputer-based method of determining a probability that a human testsubject has colorectal cancer as opposed to not having colorectalcancer, from test data representing a level of RNA encoded by a ANXA3gene in blood of the test subject, the method comprisingcomputer-implemented steps of: applying to the test data a mathematicalmodel derived from positive control data representing levels of RNAencoded by the gene in blood of human control subjects having colorectalcancer, and from negative control data representing levels of RNAencoded by the gene in blood of human control subjects not havingcolorectal cancer, wherein the mathematical model is for determining aprobability that data representing a level of RNA encoded by the genecorresponds to the positive control data and not to the negative controldata; and (b) outputting the probability that data representing a levelof RNA encoded by the gene corresponds to the positive control data andnot to the negative control data, wherein the probability that the testdata corresponds to the positive control data and not to the negativecontrol data represents the probability that the test subject hascolorectal cancer as opposed to not having colorectal cancer.

According to another aspect of the present invention there is provided amethod of classifying a human test subject as more likely to havecolorectal cancer than to not have colorectal cancer, the methodcomprising: (a) determining a level of RNA encoded by a ANXA3 gene inblood of the test subject, thereby generating test data; (b) providingnegative control data representing a level of RNA encoded by the gene inblood of human control subjects not having colorectal cancer; and (c)applying to the test data and to the negative control data amathematical formula for generating a value indicating whether the levelof RNA encoded by the gene in blood of the test subject is higher thanthe level of RNA encoded by the gene in blood of human control subjectsnot having colorectal cancer, wherein an indication by the value thatthe level of RNA encoded by the gene in blood of the test subject ishigher than the level of RNA encoded by the gene in blood of humancontrol subjects not having colorectal cancer classifies the testsubject as more likely to have colorectal cancer than to not havecolorectal cancer.

According to another aspect of the present invention there is provided amethod of classifying a human test subject as more likely to havecolorectal cancer than to not have colorectal cancer, the methodcomprising: (a) determining a level of RNA encoded by a CLEC4D gene inblood of the test subject, thereby generating test data; (b) providingnegative control data representing a level of RNA encoded by the gene inblood of human control subjects not having colorectal cancer; and (c)applying to the test data and to the negative control data amathematical formula for generating a value indicating whether the levelof RNA encoded by the gene in blood of the test subject is higher thanthe level of RNA encoded by the gene in blood of human control subjectsnot having colorectal cancer, wherein an indication by the value thatthe level of RNA encoded by the gene in blood of the test subject ishigher than the level of RNA encoded by the gene in blood of humancontrol subjects not having colorectal cancer classifies the testsubject as more likely to have colorectal cancer than to not havecolorectal cancer.

According to yet another aspect of the present invention there isprovided a method of classifying a human test subject as more likely tohave colorectal cancer than to not have colorectal cancer, the methodcomprising: (a) determining a level of RNA encoded by a IL2RB gene inblood of the test subject, thereby generating test data; (b) providingnegative control data representing a level of RNA encoded by the gene inblood of human control subjects not having colorectal cancer; and (c)applying to the test data and to the negative control data amathematical formula for generating a value indicating whether the levelof RNA encoded by the gene in blood of the test subject is higher thanthe level of RNA encoded by the gene in blood of human control subjectsnot having colorectal cancer, wherein an indication by the value thatthe level of RNA encoded by the gene in blood of the test subject islower than the level of RNA encoded by the gene in blood of humancontrol subjects not having colorectal cancer classifies the testsubject as more likely to have colorectal cancer than to not havecolorectal cancer.

According to still another aspect of the present invention there isprovided a method of classifying a human test subject as more likely tohave colorectal cancer than to not have colorectal cancer, the methodcomprising: (a) determining a level of RNA encoded by a LMNB1 gene inblood of the test subject, thereby generating test data; (b) providingnegative control data representing a level of RNA encoded by the gene inblood of human control subjects not having colorectal cancer; and (c)applying to the test data and to the negative control data amathematical formula for generating a value indicating whether the levelof RNA encoded by the gene in blood of the test subject is higher thanthe level of RNA encoded by the gene in blood of human control subjectsnot having colorectal cancer, wherein an indication by the value thatthe level of RNA encoded by the gene in blood of the test subject ishigher than the level of RNA encoded by the gene in blood of humancontrol subjects not having colorectal cancer classifies the testsubject as more likely to have colorectal cancer than to not havecolorectal cancer.

According to a further aspect of the present invention there is provideda method of classifying a human test subject as more likely to havecolorectal cancer than to not have colorectal cancer, the methodcomprising: (a) determining a level of RNA encoded by a PRRG4 gene inblood of the test subject, thereby generating test data; (b) providingnegative control data representing a level of RNA encoded by the gene inblood of human control subjects not having colorectal cancer; and (c)applying to the test data and to the negative control data amathematical formula for generating a value indicating whether the levelof RNA encoded by the gene in blood of the test subject is higher thanthe level of RNA encoded by the gene in blood of human control subjectsnot having colorectal cancer, wherein an indication by the value thatthe level of RNA encoded by the gene in blood of the test subject ishigher than the level of RNA encoded by the gene in blood of humancontrol subjects not having colorectal cancer classifies the testsubject as more likely to have colorectal cancer than to not havecolorectal cancer.

According to yet a further aspect of the present invention there isprovided a method of classifying a human test subject as more likely tohave colorectal cancer than to not have colorectal cancer, the methodcomprising: (a) determining a level of RNA encoded by a TNFAIP6 gene inblood of the test subject, thereby generating test data; (b) providingnegative control data representing a level of RNA encoded by the gene inblood of human control subjects not having colorectal cancer; and (c)applying to the test data and to the negative control data amathematical formula for generating a value indicating whether the levelof RNA encoded by the gene in blood of the test subject is higher thanthe level of RNA encoded by the gene in blood of human control subjectsnot having colorectal cancer, wherein an indication by the value thatthe level of RNA encoded by the gene in blood of the test subject ishigher than the level of RNA encoded by the gene in blood of humancontrol subjects not having colorectal cancer classifies the testsubject as more likely to have colorectal cancer than to not havecolorectal cancer.

According to still a further aspect of the present invention there isprovided a method of classifying a human test subject as more likely tohave colorectal cancer than to not have colorectal cancer, the methodcomprising: (a) determining a level of RNA encoded by a VNN1 gene inblood of the test subject, thereby generating test data; (b) providingnegative control data representing a level of RNA encoded by the gene inblood of human control subjects not having colorectal cancer; and (c)applying to the test data and to the negative control data amathematical formula for generating a value indicating whether the levelof RNA encoded by the gene in blood of the test subject is higher thanthe level of RNA encoded by the gene in blood of human control subjectsnot having colorectal cancer, wherein an indication by the value thatthe level of RNA encoded by the gene in blood of the test subject ishigher than the level of RNA encoded by the gene in blood of humancontrol subjects not having colorectal cancer classifies the testsubject as more likely to have colorectal cancer than to not havecolorectal cancer.

According to an additional aspect of the present invention there isprovided a computer-based method of classifying a human test subject asmore likely to have colorectal cancer than to not have colorectalcancer, the method comprising computer-implemented steps of: (a)applying to test data representing a level of RNA encoded by a ANXA3gene in blood of the test subject, and to negative control datarepresenting a level of RNA encoded by the gene in blood of humancontrol subjects not having colorectal cancer, a mathematical formulafor generating a value indicating whether the level of RNA encoded bythe gene in blood of the test subject is higher than the level of RNAencoded by the gene in blood of human control subjects not havingcolorectal cancer; and (b) outputting the value, wherein an indicationby the value that the level of RNA encoded by the gene in blood of thetest subject is higher than the level of RNA encoded by the gene inblood of human control subjects not having colorectal cancer classifiesthe test subject as more likely to have colorectal cancer than to nothave colorectal cancer.

According to yet an additional aspect of the present invention there isprovided a computer-based method of classifying a human test subject asmore likely to have colorectal cancer than to not have colorectalcancer, the method comprising computer-implemented steps of: (a)applying to test data representing a level of RNA encoded by a CLEC4Dgene in blood of the test subject, and to negative control datarepresenting a level of RNA encoded by the gene in blood of humancontrol subjects not having colorectal cancer, a mathematical formulafor generating a value indicating whether the level of RNA encoded bythe gene in blood of the test subject is higher than the level of RNAencoded by the gene in blood of human control subjects not havingcolorectal cancer; and (b) outputting the value, wherein an indicationby the value that the level of RNA encoded by the gene in blood of thetest subject is higher than the level of RNA encoded by the gene inblood of human control subjects not having colorectal cancer classifiesthe test subject as more likely to have colorectal cancer than to nothave colorectal cancer.

According to still an additional aspect of the present invention thereis provided a computer-based method of classifying a human test subjectas more likely to have colorectal cancer than to not have colorectalcancer, the method comprising computer-implemented steps of: (a)applying to test data representing a level of RNA encoded by a IL2RBgene in blood of the test subject, and to negative control datarepresenting a level of RNA encoded by the gene in blood of humancontrol subjects not having colorectal cancer, a mathematical formulafor generating a value indicating whether the level of RNA encoded bythe gene in blood of the test subject is lower than the level of RNAencoded by the gene in blood of human control subjects not havingcolorectal cancer; and (b) outputting the value, wherein an indicationby the value that the level of RNA encoded by the gene in blood of thetest subject is lower than the level of RNA encoded by the gene in bloodof human control subjects not having colorectal cancer classifies thetest subject as more likely to have colorectal cancer than to not havecolorectal cancer.

According to yet still an additional aspect of the present inventionthere is provided a computer-based method of classifying a human testsubject as more likely to have colorectal cancer than to not havecolorectal cancer, the method comprising computer-implemented steps of:(a) applying to test data representing a level of RNA encoded by a LMNB1gene in blood of the test subject, and to negative control datarepresenting a level of RNA encoded by the gene in blood of humancontrol subjects not having colorectal cancer, a mathematical formulafor generating a value indicating whether the level of RNA encoded bythe gene in blood of the test subject is higher than the level of RNAencoded by the gene in blood of human control subjects not havingcolorectal cancer; and (b) outputting the value, wherein an indicationby the value that the level of RNA encoded by the gene in blood of thetest subject is higher than the level of RNA encoded by the gene inblood of human control subjects not having colorectal cancer classifiesthe test subject as more likely to have colorectal cancer than to nothave colorectal cancer.

According to another aspect of the present invention there is provided acomputer-based method of classifying a human test subject as more likelyto have colorectal cancer than to not have colorectal cancer, the methodcomprising computer-implemented steps of: (a) applying to test datarepresenting a level of RNA encoded by a PRRG4 gene in blood of the testsubject, and to negative control data representing a level of RNAencoded by the gene in blood of human control subjects not havingcolorectal cancer, a mathematical formula for generating a valueindicating whether the level of RNA encoded by the gene in blood of thetest subject is higher than the level of RNA encoded by the gene inblood of human control subjects not having colorectal cancer; and (b)outputting the value, wherein an indication by the value that the levelof RNA encoded by the gene in blood of the test subject is higher thanthe level of RNA encoded by the gene in blood of human control subjectsnot having colorectal cancer classifies the test subject as more likelyto have colorectal cancer than to not have colorectal cancer. Accordingto yet another aspect of the present invention there is provided acomputer-based method of classifying a human test subject as more likelyto have colorectal cancer than to not have colorectal cancer, the methodcomprising computer-implemented steps of: (a) applying to test datarepresenting a level of RNA encoded by a TNFAIP6 gene in blood of thetest subject, and to negative control data representing a level of RNAencoded by the gene in blood of human control subjects not havingcolorectal cancer, a mathematical formula for generating a valueindicating whether the level of RNA encoded by the gene in blood of thetest subject is higher than the level of RNA encoded by the gene inblood of human control subjects not having colorectal cancer; and (b)outputting the value, wherein an indication by the value that the levelof RNA encoded by the gene in blood of the test subject is higher thanthe level of RNA encoded by the gene in blood of human control subjectsnot having colorectal cancer classifies the test subject as more likelyto have colorectal cancer than to not have colorectal cancer.

According to still another aspect of the present invention there isprovided a computer-based method of classifying a human test subject asmore likely to have colorectal cancer than to not have colorectalcancer, the method comprising computer-implemented steps of: (a)applying to test data representing a level of RNA encoded by a VNN1 genein blood of the test subject, and to negative control data representinga level of RNA encoded by the gene in blood of human control subjectsnot having colorectal cancer, a mathematical formula for generating avalue indicating whether the level of RNA encoded by the gene in bloodof the test subject is higher than the level of RNA encoded by the genein blood of human control subjects not having colorectal cancer; and (b)outputting the value, wherein an indication by the value that the levelof RNA encoded by the gene in blood of the test subject is higher thanthe level of RNA encoded by the gene in blood of human control subjectsnot having colorectal cancer classifies the test subject as more likelyto have colorectal cancer than to not have colorectal cancer.

According to a further aspect of the present invention there is provideda method of classifying a human test subject as more likely to havecolorectal cancer than to not have colorectal cancer, the methodcomprising, for each gene of a set of one or more genes selected fromthe group consisting of ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 andVNN1: (a) determining a level of RNA encoded by the gene in blood of thetest subject, thereby generating test data; (b) providing negativecontrol data representing levels of RNA encoded by the gene in blood ofhuman control subjects not having colorectal cancer; and (c) applying tothe test data and to the negative control dataa mathematical formula forgenerating a value indicating, for ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6and VNN1, whether the level of RNA encoded by the gene in blood of thetest subject is higher than the level of RNA encoded by the gene inblood of human control subjects not having colorectal cancer, andindicating, for IL2RB, whether the level of RNA encoded by the gene inblood of the test subject is lower than the level of RNA encoded by thegene in blood of human control subjects not having colorectal cancer,wherein, for ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1, anindication by the value that the level of RNA encoded by the gene inblood of the test subject is higher than the level of RNA encoded by thegene in blood of human control subjects not having colorectal cancerclassifies the test subject as more likely to have colorectal cancerthan to not have colorectal cancer, and wherein, for IL2RB, anindication by the value that the level of RNA encoded by the gene inblood of the test subject is lower than the level of RNA encoded by thegene in blood of human control subjects not having colorectal cancerclassifies the test subject as more likely to have colorectal cancerthan to not have colorectal cancer.

According to yet a further aspect of the present invention there isprovided a computer-based method of classifying a human test subject asmore likely to have colorectal cancer than to not have colorectalcancer, the method comprising, for each gene of a set of one or moregenes selected from the group consisting of ANXA3, CLEC4D, IL2RB, LMNB1,PRRG4, TNFAIP6 and VNN1, computer-implemented steps of: applying to testdata representing a level of RNA encoded by the gene in blood of thetest subject, and to negative control data representing a level of RNAencoded by the gene in blood of human control subjects not havingcolorectal cancer, a formula for calculating a value indicating, forANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1, whether the level of RNAencoded by the gene in blood of the test subject is higher than thelevel of RNA encoded by the gene in blood of human control subjects nothaving colorectal cancer, and indicating, for IL2RB, whether the levelof RNA encoded by the gene in blood of the test subject is lower thanthe level of RNA encoded by the gene in blood of human control subjectsnot having colorectal cancer, wherein, for ANXA3, CLEC4D, LMNB1, PRRG4,TNFAIP6 and VNN1, an indication by the value that the level of RNAencoded by the gene in blood of the test subject is higher than thelevel of RNA encoded by the gene in blood of human control subjects nothaving colorectal cancer classifies the test subject as more likely tohave colorectal cancer than to not have colorectal cancer, and wherein,for IL2RB, an indication by the value that the level of RNA encoded bythe gene in blood of the test subject is lower than the level of RNAencoded by the gene in blood of human control subjects not havingcolorectal cancer classifies the test subject as more likely to havecolorectal cancer than to not have colorectal cancer.

According to one aspect of the invention there is provided a method ofdiagnosing colorectal cancer in a test subject, the method comprising,for each gene of a set of one or more genes selected from the groupconsisting of ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1: (a)determining a level of RNA encoded by the gene in blood of the testsubject, thereby generating test data; (b) providing positive controldata representing levels of RNA encoded by the gene in blood of humancontrol subjects having colorectal cancer, and providing negativecontrol data representing levels of RNA encoded by the gene in blood ofhuman control subjects not having colorectal cancer; and (c) determininga probability that the test data corresponds to the positive controldata and not to the negative control data, wherein a determination thatthe test data corresponds to the positive control data and not to thenegative control data provides an indication of colorectal cancer in thetest subject.

According to a further aspect of the present invention there is provideda method of diagnosing colorectal cancer in a test subject, the methodcomprising, for each gene of a set of one or more genes selected fromthe group consisting of ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 andVNN1: (a) determining a level of RNA encoded by the gene in blood of thetest subject, thereby generating test data; (b) providing negativecontrol data representing levels of RNA encoded by the gene in blood ofhuman control subjects not having colorectal cancer; and (c) applying tothe test data and to the negative control data a mathematical formulafor generating a value indicating, for ANXA3, CLEC4D, LMNB1, PRRG4,TNFAIP6 and VNN1, whether the level of RNA encoded by the gene in bloodof the test subject is higher than the level of RNA encoded by the genein blood of human control subjects not having colorectal cancer, andindicating, for IL2RB, whether the level of RNA encoded by the gene inblood of the test subject is lower than the level of RNA encoded by thegene in blood of human control subjects not having colorectal cancer,wherein, for ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1, anindication by the value that the level of RNA encoded by the gene inblood of the test subject is higher than the level of RNA encoded by thegene in blood of human control subjects not having colorectal cancerprovides an indication of colorectal cancer in the test subject, andwherein, for IL2RB, an indication by the value that the level of RNAencoded by the gene in blood of the test subject is lower than the levelof RNA encoded by the gene in blood of human control subjects not havingcolorectal cancer provides an indication of colorectal cancer in thetest subject.

According to another aspect of the present invention there is provided amethod of determining whether a test subject is at an increased risk ofhaving colorectal cancer relative to the general population, comprising:a) obtaining a test sample of blood from the subject; and i) determininga level of RNA encoded by a annexin A3 (ANXA3) gene in the test sampleof blood, ii) comparing the level of RNA encoded by the gene asdetermined in step (i) with the level of the RNA encoded by the gene incontrol samples of blood; and b) concluding that the subject is at anincreased risk of having colorectal cancer relative to the generalpopulation if the level of RNA encoded by the gene in the test sample ofblood is higher than in the control samples of blood.

According to yet another aspect of the present invention there isprovided a method of determining whether a test subject is at anincreased risk of having colorectal cancer relative to the generalpopulation, comprising: a) obtaining a test sample of blood from thesubject; and i) determining a level of RNA encoded by a C-type lectindomain family 4, member D (CLEC4D) gene in the test sample of blood, ii)comparing the level of RNA encoded by the gene as determined in step (i)with the level of the RNA encoded by the gene in control samples ofblood; and b) concluding that the subject is at an increased risk ofhaving colorectal cancer relative to the general population if the levelof RNA encoded by the gene in the test sample of blood is higher than inthe control samples of blood.

According to still another aspect of the present invention there isprovided a method of determining whether a test subject is at anincreased risk of having colorectal cancer relative to the generalpopulation, comprising: a) obtaining a test sample of blood from thesubject; and i) determining a level of RNA encoded by a interleukin 2receptor, beta (IL2RB) gene in the test sample of blood, ii) comparingthe level of RNA encoded by the gene as determined in step (i) with thelevel of the RNA encoded by the gene in control samples of blood; and b)concluding that the subject is at an increased risk of having colorectalcancer relative to the general population if the level of RNA encoded bythe gene in the test sample of blood is lower than in the controlsamples of blood.

According to a further aspect of the present invention there is provideda method of determining whether a test subject is at an increased riskof having colorectal cancer relative to the general population,comprising: a) obtaining a test sample of blood from the subject; and i)determining a level of RNA encoded by a lamin B1 (LMNB1) gene in thetest sample of blood, ii) comparing the level of RNA encoded by the geneas determined in step (i) with the level of the RNA encoded by the genein control samples of blood; and b) concluding that the subject is at anincreased risk of having colorectal cancer relative to the generalpopulation if the level of RNA encoded by the gene in the test sample ofblood is higher than in the control samples of blood.

According to yet a further aspect of the present invention there isprovided a method of determining whether a test subject is at anincreased risk of having colorectal cancer relative to the generalpopulation, comprising: a) obtaining a test sample of blood from thesubject; and i) determining a level of RNA encoded by a proline rich Gla(G carboxyglutamic acid) 4 (transmembrane) (PRRG4) gene in the testsample of blood, ii) comparing the level of RNA encoded by the gene asdetermined in step (i) with the level of the RNA encoded by the gene incontrol samples of blood; and b) concluding that the subject is at anincreased risk of having colorectal cancer relative to the generalpopulation if the level of RNA encoded by the gene in the test sample ofblood is higher than in the control samples of blood.

According to still a further aspect of the present invention there isprovided a method of determining whether a test subject is at anincreased risk of having colorectal cancer relative to the generalpopulation, comprising: a) obtaining a test sample of blood from thesubject; and i) determining a level of RNA encoded by a tumor necrosisfactor, alpha induced protein 6 (TNFAIP6) gene in the test sample ofblood, ii) comparing the level of RNA encoded by as determined in step(i) with the level of the RNA encoded by the gene in control samples ofblood; and b) concluding that the subject is at an increased risk ofhaving colorectal cancer relative to the general population if the levelof RNA encoded by the gene in the test sample of blood is higher than inthe control samples of blood.

According to still a further aspect of the present invention there isprovided a method of determining whether a test subject is at anincreased risk of having colorectal cancer relative to the generalpopulation, comprising: a) obtaining a test sample of blood from thesubject; and i) determining a level of RNA encoded by a vanin 1 (VNN1)gene in the test sample of blood, ii) comparing the level of RNA encodedby the gene as determined in step (i) with the level of the RNA encodedby the gene in control samples of blood; and b) concluding that thesubject is at an increased risk of having colorectal cancer relative tothe general population if the level of RNA encoded by the gene in thetest sample of blood is higher than in the control samples of blood.

According to an additional aspect of the present invention there isprovided a method of determining whether a test subject is at anincreased risk of having colorectal cancer relative to the generalpopulation, comprising: a) obtaining a test sample of blood from thesubject; and for each gene of a set of genes selected from the groupconsisting of: ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1, i)determining a level of RNA encoded by the gene in the test sample ofblood, ii) comparing the level of RNA encoded by the gene of the set asdetermined in step (i) with the level of RNA encoded by the gene in oneor more control samples of blood; and b) concluding that the subject isat an increased risk of having colorectal cancer relative to the generalpopulation if, for ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1, thelevel of RNA encoded by the gene in the test sample of blood is higherthan in the control samples of blood, and concluding that the subject isat an increased risk of having colorectal cancer relative to the generalpopulation if, for IL2RB, the level of RNA encoded by the gene in thetest sample of blood is lower than in the control samples of blood.

According to an additional aspect of the present invention there isprovided a method of diagnosing colorectal cancer in a test subject,comprising: a) obtaining a test sample of blood from the subject; andfor each gene of a set of genes selected from the group consisting of:ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1, i) determining alevel of RNA encoded by the gene in the test sample of blood, and ii)applying to the level of RNA encoded by the gene of the set asdetermined in step (i) and to the level of RNA encoded by the gene inone or more control samples of blood a mathematical formula forgenerating a value indicating whether, for ANXA3, CLEC4D, LMNB1, PRRG4,TNFAIP6 and VNN1, the level of RNA encoded by the gene in the testsample of blood is higher than in the control samples of blood, and, forIL2RB, the level of RNA encoded by the gene in the test sample of bloodis lower than in the control samples of blood; and b) concluding thatthere is an indication of colorectal cancer in the test subject, if, forANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1, the value indicates thatthe level of RNA encoded by the gene in the test sample of blood ishigher than in the control samples of blood, and concluding that thereis an indication of colorectal cancer in the test subject if, for IL2RB,the value indicates that the level of RNA encoded by the gene in thetest sample of blood is lower than in the control samples of blood.

According to further features of the invention described below, thecontrol samples are from individuals who have been diagnosed as nothaving colorectal cancer.

According to still another aspect of the invention there is provided akit comprising packaging and containing, for each gene of a set of twoor more genes selected from the group consisting of ACTB, ANXA3, CLEC4D,IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1, a primer set capable ofgenerating an amplification product of a polynucleotide complementary toRNA encoded, in a human subject, only by the gene.

According to further features of the invention described below, the kitfurther contains two or more components selected from the groupconsisting of a thermostable polymerase, a reverse transcriptase,deoxynucleotide triphosphates, nucleotide triphosphates and enzymebuffer.

According to further features of the invention described below, the kitfurther contains at least one labeled probe capable of selectivelyhybridizing to either a sense or an antisense strand of theamplification product.

According to further features of the invention described below, the kitfurther contains a computer-readable medium having instructions storedthereon that are operable when executed by a computer for applying amathematical model to test data representing a level of RNA encoded bythe gene in blood of a human test subject, wherein the mathematicalmodel is derived from positive control data representing levels of RNAencoded by the gene in blood of human control subjects having colorectalcancer, and from negative control data representing levels of RNAencoded by the gene in blood of human control subjects not havingcolorectal cancer, wherein the mathematical model is for determining aprobability that data representing a level of RNA encoded by the genecorresponds to the positive control data and not to the negative controldata, and wherein the probability that the test data corresponds to thepositive control data and not to the negative control data representsthe probability that the test subject has colorectal cancer as opposedto not having colorectal cancer.

According to further features of the invention described below, the kitfurther contains a computer-readable medium having instructions storedthereon that are operable when executed by a computer for applying, totest data representing a level of RNA encoded by the gene in blood of ahuman test subject, and to negative control data representing a level ofRNA encoded by the gene in blood of human control subjects not havingcolorectal cancer, a mathematical formula for generating a valueindicating, for ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1, whetherthe level of RNA encoded by the gene in blood of the test subject ishigher than the level of RNA encoded by the gene in blood of humancontrol subjects not having colorectal cancer, and, for IL2RB, whetherthe level of RNA encoded by the gene in blood of the test subject islower than the level of RNA encoded by the gene in blood of humancontrol subjects not having colorectal cancer, wherein, for ANXA3,CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1, an indication by the value thatthe level of RNA encoded by the gene in blood of the test subject ishigher than the level of RNA encoded by the gene in blood of humancontrol subjects not having colorectal cancer classifies the testsubject as more likely to have colorectal cancer than to not havecolorectal cancer, and wherein, for IL2RB, an indication by the valuethat the level of RNA encoded by the gene in blood of the test subjectis lower than the level of RNA encoded by the gene in blood of humancontrol subjects not having colorectal cancer classifies the testsubject as more likely to have colorectal cancer than to not havecolorectal cancer.

According to further features of the invention described below, the setof one or more genes consists of ACTB and one or more genes selectedfrom the group consisting of ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6and VNN1.

According to further features of the invention described below, the setof one or more genes consists of ACTB and ANXA3.

According to further features of the invention described below, the setof one or more genes consists of ACTB and CLEC4D.

According to further features of the invention described below, the setof one or more genes consists of ACTB and IL2RB.

According to further features of the invention described below, the setof one or more genes consists of ACTB and LMNB1.

According to further features of the invention described below, the setof one or more genes consists of ACTB and PRRG4.

According to further features of the invention described below, the setof one or more genes consists of TNFAIP6 and PRRG4.

According to further features of the invention described below, the setof one or more genes consists of ACTB and VNN1.

According to further features of the invention described below, thelevel of RNA encoded by the gene in blood of the test subject isdetermined via quantitative reverse transcriptase-polymerase chainreaction analysis.

According to further features of the invention described below, thelevel of RNA encoded by the gene in blood of the test subject and thelevels of RNA encoded by the gene in blood of the control subjects aredetermined via the same method.

According to further features of the invention described below, the setof one or more genes is a set of one or more genes selected from thegroup consisting of ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 andVNN1, wherein the level of RNA encoded by the gene in blood of the testsubject is determined as a ratio to a level of RNA encoded by ACTB inblood of the test subject.

According to further features of the invention described below, thelevel of RNA encoded by the gene in blood of the test subject and thelevel of RNA encoded by ACTB in blood of the test subject are determinedvia duplex quantitative reverse transcriptase-polymerase chain reactionanalysis of RNA encoded by the gene and of RNA encoded by ACTB.

According to further features of the invention described below, the setof one or more genes consists of IL2RB and one or more genes selectedfrom the group consisting of ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 andVNN1.

According to further features of the invention described below, the setof one or more genes is a set of one or more genes selected from thegroup consisting of ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1, andwherein the level of RNA encoded by the gene in blood of the testsubject is determined as a ratio to a level of RNA encoded by IL2RB inblood of the test subject.

According to further features of the invention described below, thelevel of RNA encoded by the gene in blood of the test subject and thelevel of RNA encoded by IL2RB in blood of the test subject aredetermined via duplex quantitative reverse transcriptase-polymerasechain reaction analysis of RNA encoded by the gene and of RNA encoded byIL2RB.

According to further features of the invention described below, the setof one or more genes consists of ANXA3.

According to further features of the invention described below, the setof one or more genes consists of CLEC4D.

According to further features of the invention described below, the setof one or more genes consists of IL2RB.

According to further features of the invention described below, the setof one or more genes consists of LMNB1.

According to further features of the invention described below, the setof one or more genes consists of PRRG4.

According to further features of the invention described below, the setof one or more genes consists of TNFAIP6.

According to further features of the invention described below, the setof one or more genes consists of VNN1.

According to further features of the invention described below, the setof one or more genes consists of IL2RB and ANXA3.

According to further features of the invention described below, the setof one or more genes consists of IL2RB and CLEC4D.

According to further features of the invention described below, the setof one or more genes consists of IL2RB and LMNB1.

According to further features of the invention described below, the setof one or more genes consists of IL2RB and PRRG4.

According to further features of the invention described below, the setof one or more genes consists of IL2RB and TNFAIP6.

According to further features of the invention described below, the setof one or more genes consists of IL2RB and VNN1.

DEFINITIONS

As will become apparent, preferred features and characteristics of oneaspect of the invention are applicable to any other aspect of theinvention. It should be noted that, as used herein, the singular form“a”, “an” and “the” include plural references unless the context clearlydictates otherwise.

“Encode” A polynucleotide, including a gene, is said the to “encode” aRNA and/or polypeptide if, in its native state or when manipulated bymethods well known to those skilled in the art, it can be transcribedand/or translated to produce the mRNA for and/or the polypeptide or afragment thereof. The anti-sense strand is the complement of such anucleic acid, and the encoding sequence can be deduced there from.

The term “label” refers to a composition capable of producing adetectable signal indicative of the presence of the targetpolynucleotide in an assay sample. Suitable labels includeradioisotopes, nucleotide chromophores, enzymes, substrates, fluorescentmolecules, chemiluminescent moieties, magnetic particles, bioluminescentmoieties, and the like. As such, a label is any composition detectableby spectroscopic, photochemical, biochemical, immunochemical,electrical, optical or chemical means.

As used herein, a “sample” refers to a sample of tissue or fluidisolated from an individual, including but not limited to, for example,blood, plasma, serum, tumor biopsy, urine, stool, sputum, spinal fluid,pleural fluid, nipple aspirates, lymph fluid, the external sections ofthe skin, respiratory, intestinal, and genitourinary tracts, tears,saliva, milk, cells (including but not limited to blood cells), organs,and also samples of in vitro cell culture constituent.

Examples of amplification techniques include strand displacementamplification, as disclosed in U.S. Pat. No. 5,744,311;transcription-free isothermal amplification, as disclosed in U.S. Pat.No. 6,033,881; repair chain reaction amplification, as disclosed in WO90/01069; ligase chain reaction amplification, as disclosed in EuropeanPatent Appl. 320 308; gap filling ligase chain reaction amplification,as disclosed in U.S. Pat. No. 5,427,930; and RNA transcription-freeamplification, as disclosed in U.S. Pat. No. 6,025,134.

Examples of a primer of the invention include an oligonucleotide whichis capable of acting as a point of initiation of polynucleotidesynthesis along a complementary strand when placed under conditions inwhich synthesis of a primer extension product which is complementary toa polynucleotide is catalyzed. Such conditions include the presence offour different nucleotide triphosphates or nucleoside analogs and one ormore agents for polymerization such as DNA polymerase and/or reversetranscriptase, in an appropriate buffer (“buffer” includes substituentswhich are cofactors, or which affect pH, ionic strength, etc.), and at asuitable temperature. A primer must be sufficiently long to prime thesynthesis of extension products in the presence of an agent forpolymerase. A typical primer contains at least about 5 nucleotides inlength of a sequence substantially complementary to the target sequence,but somewhat longer primers are preferred.

The terms “complementary” or “complement thereof”, as used herein, referto sequences of polynucleotides which are capable of forming Watson &Crick base pairing with another specified polynucleotide throughout theentirety of the complementary region. This term is applied to pairs ofpolynucleotides based solely upon their sequences and does not refer toany specific conditions under which the two polynucleotides wouldactually bind.

A primer will always contain a sequence substantially complementary tothe target sequence, that is the specific sequence to be amplified, towhich it can anneal.

In the context of this invention, the term “probe” refers to a moleculewhich can detectably distinguish between target molecules differing instructure, such as allelic variants. Detection can be accomplished in avariety of different ways but preferably is based on detection ofspecific binding. Examples of such specific binding include antibodybinding and nucleic acid probe hybridization.

The term “gene” as used herein is a polynucleotide which may includecoding sequences, intervening sequences and regulatory elementscontrolling transcription and/or translation. Genes of the inventioninclude normal alleles of the gene encoding polymorphisms, includingsilent alleles having no effect on the amino acid sequence of the gene'sencoded polypeptide as well as alleles leading to amino acid sequencevariants of the encoded polypeptide that do not substantially affect itsfunction. These terms also may otpyiosnlly include alleles having one ormore mutations which affect the function of the encoded polypeptide'sfunction.

The polynucleotide compositions, such as primers of the invention, ofthis invention include RNA, cDNA, DNA complementary to target cDNA ofthis invention or portion thereof, genomic DNA, unspliced RNA, splicedRNA, alternately spliced RNA, synthetic forms, and mixed polymers, bothsense and antisense strands, and may be chemically or biochemicallymodified or may contain non-natural or derivatized nucleotide bases, aswill be readily appreciated by those skilled in the art.

Where nucleic acid according to the invention includes RNA, reference tothe sequence shown should be construed as reference to the RNAequivalent, with U substituted for T.

The term “amount” or “level” of RNA encoded by a gene of the invention,preferably a colorectal cancer biomarker gene described herein, or ahousekeeping gene, encompasses the absolute amount of the RNA, therelative amount or concentration of the RNA, as well as any value orparameter which correlates thereto.

The methods of nucleic acid isolation, amplification and analysis areroutine for one skilled in the art and examples of protocols can befound, for example, in the Molecular Cloning: A Laboratory Manual(3-Volume Set) Ed. Joseph Sambrook, David W. Russel, and Joe Sambrook,Cold Spring Harbor Laboratory; 3rd edition (Jan. 15, 2001), ISBN:0879695773. Particularly useful protocol source for methods used in PCRamplification is PCR (Basics: From Background to Bench) by M. J.McPherson, S. G. Moller, R. Beynon, C. Howe, Springer Verlag; 1stedition (Oct. 15, 2000), ISBN: 0387916008.

“Kit” refers to a combination of physical elements, e.g., probes,including without limitation specific primers, labeled nucleic acidprobes, antibodies, protein-capture agent(s), reagent(s), instructionsheet(s) and other elements useful to practice the invention, inparticular to identify the levels of particular RNA molecules in asample. These physical elements can be arranged in any way suitable forcarrying out the invention. For example, probes and/or primers can beprovided in one or more containers or in an array or microarray device.

Colorectal cancer, also called colon cancer or rectal cancer orcolorectal carcinoma, is cancer that forms in either the colon or therectum.

The present invention is useful in a diagnostic product or method todetect the level of RNA of genes of interest, in particular, thecolorectal biomarkers of the present invention. Accordingly, theinvention encompasses the use of diagnostic kits based on a variety ofmethodologies, e.g., PCR, reverse transcriptase-PCR, quantitative PCR,microarray, chip, mass-spectroscopy, which are capable of detecting RNAlevels in a sample. The invention also provides an article ofmanufacturing comprising packaging material and an analytical agentcontained within the packaging material, wherein the analytical agentcan be used for determining and/or comparing the levels of RNA encodedby one or more target genes of the invention, and wherein the packagingmaterial comprises a label or package insert which indicates that theanalytical agent can be used to identify levels of RNA that correspondto a probability that a test subject has colorectal cancer, such as aprobability that the test subject has colorectal cancer as opposed tonot having colorectal cancer.

The present invention therefore provides kits comprising degenerateprimers to amplify polymorphic alleles or variants of target genes ofthe invention, and instructions comprising an amplification protocol andanalysis of the results. The kit may alternatively also comprisebuffers, enzymes, and containers for performing the amplification andanalysis of the amplification products. The kit may also be a componentof a screening or prognostic kit comprising other tools such as DNAmicro arrays. The kit may also provides one or more control templates,such as nucleic acids isolated from sample of patientss withoutcolorectal cancer, and/or nucleic acids isolated from ssamples ofpatients with colorectal cancer.

The kit may also include instructions for use of the kit to amplifyspecific targets on a solid support. Where the kit contains a preparedsolid support having a set of primers already fixed on the solidsupport, e.g. for amplifying a particular set of target polynucleotides,the kit also includes reagents necessary for conducting a PCR on a solidsupport, for example using an in situ-type or solid phase type PCRprocedure where the support is capable of PCR amplification using an insitu-type PCR machine. The PCR reagents, included in the kit, includethe usual PCR buffers, a thermostable polymerase (e.g. Taq DNApolymerase), nucleotides (e.g. dNTPs), and other components and labelingmolecules (e.g. for direct or indirect labeling). The kits can beassembled to support practice of the PCR amplification method usingimmobilized primers alone or, alternatively, together with solutionphase primers.

In one embodiment, the kit provides one or more primer pairs, each paircapable of amplifying RNA encoded by a target gene of the invention,thereby providing a kit for analysis of RNA expression of severaldifferent target genes of the invention in a biological sample in onereaction or several parallel reactions. Primers in the kits may belabeled, for example fluorescently labeled, to facilitate detection ofthe amplification products and consequent analysis of the RNA levels.

In one embodiment, levels of RNA encoded by more than one target genecan be determined in one analysis. A combination kit may thereforeinclude primers capable of amplifying cDNA derived from RNA encoded bydifferent target genes. The primers may be differentially labeled, forexample using different fluorescent labels, so as to differentiatebetween RNA from different target genes.

Multiplex, such as duplex, real-time RT-PCR enables simultaneousquantification of 2 targets in the same reaction, which saves time,reduces costs, and conserves samples. These advantages of multiplex,real-time RT-PCR make the technique well-suited for high-throughput geneexpression analysis. Multiplex qPCR assay in a real-time formatfacilitates quantitative measurements and minimizes the risk offalse-negative results. It is essential that multiplex PCR is optimizedso that amplicons of all samples are compared insub-plateau phase ofPCR. Yun, Z., I. Lewensohn-Fuchs, P. Ljungman, L. Ringholm, J. Jonsson,and J. Albert. 2003. A real-time TaqMan PCR for routine quantitation ofcytomegalovirus DNA in crude leukocyte lysates from stem cell transplantpatients. J. Viol. Methods 110:73-79. [PubMed]. Yun, Z., I.Lewensohn-Fuchs, P. Ljungman, and A. Vahlne 2000. Real-time monitoringof cytomegalovirus infections after stem cell transplantation using theTaqMan polymerase chain reaction assays. Transplantation 69:1733-1736.[PubMed]. Simultaneous quantification of up to 2, 3, 4, 5, 6, 7, and 8or more targets may be useful.

The primers and probes contained within the kit may include those listedin 19, and various subcombinations thereof.

A “control population” refers to a defined group of individuals or agroup of individuals with or without colorectal cancer, and mayoptionally be further identified by, but not limited to geographic,ethnic, race, gender, one or more other conditions or diseases, and/orcultural indices. In most cases a control population may encompass atleast 10, 50, 100, 1000, or more individuals.

“Positive control data” encompasses data representing levels of RNAencoded by a target gene of the invention in each of one or moresubjects having colorectal cancer of the invention, and encompasses asingle data point representing an average level of RNA encoded by atarget gene of the invention in a plurality of subjects havingcolorectal cancer of the invention.

“Negative control data” encompasses data representing levels of RNAencoded by a target gene of the invention in each of one or moresubjects not having colorectal cancer of the invention, and encompassesa single data point representing an average level of RNA encoded by atarget gene of the invention in a plurality of subjects havingcolorectal cancer of the invention.

The probability that test data of the invention “corresponds” topositive control data or negative control data of the invention refersto the probability that the test data is more likely to becharacteristic of data obtained in subjects having colorectal cancerthan in subjects not having any colorectal pathology, or is more likelyto be characteristic of data obtained in subjects not having anycolorectal pathology than in subjects having colorectal cancer,respectively.

A primer which “selectively hybridizes” to a target polynucleotide is aprimer which is capable of hybridizing only, or mostly, with a singletarget polynucleotide in a mixture of polynucleotides consisting of RNAof human blood, or consisting of DNA complementary to RNA of humanblood.

A gene expression profile of the invention for colorectal cancer foundin blood at the RNA level of one or more genes comprising, butpreferably not limited to, an ANXA3 gene, a CLEC4D gene, an IL2RB gene,an LMNB1 gene, a PRRG4 gene, a TNFAIP6 gene and a VNN1 gene, can beidentified or confirmed using many techniques, including but preferablynot limited to PCR methods, as for example discussed further in theworking examples herein, Northern analyses and and the microarraytechnique. This gene expression profile can be measured in a bodilysample, such as blood, using microarray technology. In an embodiment ofthis method, fluorescently labeled cDNA probes may be generated throughincorporation of fluorescent nucleotides by reverse transcription of RNAextracted from blood. Labeled cDNA probes applied to the chip hybridizewith specificity to each spot of DNA on the array. Quantitation ofhybridization of each arrayed element allows for assessment ofcorresponding mRNA abundance. For example, with dual color fluorescence,separately labeled cDNA probes generated from two sources of RNA arehybridized pair wise to the array. The relative abundance of thetranscripts from the two sources corresponding to each specified gene isthus determined simultaneously. Such methods have been shown to have thesensitivity required to detect rare transcripts, which are expressed ata few copies per cell, and to reproducibly detect at least approximatelytwo-fold differences in the expression levels (Schena et al., Proc.Natl. Acad. Sci. USA 93(2):106-149 (1996)). Microarray analysis can beperformed by commercially available equipment, following manufacturer'sprotocols, such as by using the Affymetrix GenChip technology, orIncyte's micro array technology.

Other features and advantages of the invention will become apparent fromthe following detailed description. It should be understood, however,that the detailed description and the specific examples while indicatingpreferred embodiments of the invention are given by way of illustrationonly, since various changes and modifications within the spirit andscope of the invention will become apparent to those skilled in the artfrom this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described in relation to the drawings inwhich:

FIGS. 1A-H are sequence diagrams depicting the nucleotide sequences ofthe following genes: ACTB, ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6and VNN1, respectively.

FIG. 2 is a schematic depicting an exemplary computer system forpracticing certain of the methods described herein.

DETAILED DESCRIPTION

The invention is of methods, kits, computer systems andcomputer-readable media for determining a probability that a humansubject has colorectal cancer. Specifically, the invention can be usedto determine such a probability via analysis of novel markers ofcolorectal cancer in blood which are disclosed herein.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details set forth in the following description or exemplified bythe Examples. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

Effective methods of testing for colorectal cancer via analysis of bloodmarkers would overcome critical disadvantages of prior art methods,which are excessively invasive, cumbersome, risky, unavailable and/orassociated with low patient screening compliance rates. While variousapproaches have been proposed in the prior art for colorectal cancertesting via analysis of markers of this disease in blood (reviewed inHundt S. et al. Blood markers for early detection of colorectal cancer:a systematic review. Cancer Epidemiol Biomarkers Prey. 2007 October;16(10):1935-53), none of these approaches, however, has demonstrated acapacity to satisfactorily enable determination of the probability thata test subject has colorectal cancer as opposed to not having colorectalcancer.

Thus, the prior art fails to provide an effective method of testing asubject for colorectal cancer via analysis in a blood sample of levelsof RNA encoded by one or more of the genes ANXA3, CLEC4D, LMNB1, PRRG4,TNFAIP6 and VNN1 blood markers.

While reducing the invention to practice it was surprisingly uncoveredthat levels of RNA encoded by the genes ANXA3, CLEC4D, LMNB1, PRRG4,TNFAIP6 and VNN1 are significantly higher in blood of subjects havingcolorectal cancer than in blood of subjects not having any colorectalpathology, and that levels of RNA encoded by IL2RB are significantlylower in blood of subjects having colorectal cancer than in blood ofsubjects not having any colorectal pathology (Example 2). While furtherreducing the invention to practice, it was surprisingly uncovered thatmathematical models based on levels of RNA encoded by the 127 possiblecombinations of the colorectal cancer marker genes ANXA3, CLEC4D, IL2RB,LMNB1, PRRG4, TNFAIP6 and VNN1 in blood of a test subject could bederived capable of discriminating between subjects having colorectalcancer and subjects not having any colorectal pathology (Example 2).While further reducing the invention to practice, it was surprisinglyuncovered that mathematical models based on levels of RNA encoded by the63 possible combinations of the colorectal cancer marker genes ANXA3,CLEC4D, LMNB1, PRRG4, TNFAIP6, and VNN1 in blood of a test subject, whennormalized against levels of RNA encoded by IL2RB, could be derivedcapable of discriminating between subjects having colorectal cancer andsubjects not having any colorectal pathology (Example 3). It will beappreciated that application of such mathematical models to test datarepresenting blood levels in a test subject of RNA encoded by theaforementioned novel colorectal cancer marker genes disclosed herein canbe used to provide the probability that the test subject has colorectalcancer as opposed to not having any colorectal pathology.

While reducing the invention to practice, fold changes of blood levelsof RNA encoded by ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1,including fold-changes of levels normalized to IL2RB, in subjects havingcolorectal cancer relative to subjects not having any colorectalpathology were surprisingly uncovered (Example 2, Example 3 and Example6).

Thus, according to one aspect of the invention there is provided amethod of determining a probability that a human test subject hascolorectal cancer as opposed to not having colorectal cancer. In a firststep, the method is effected by determining, for each gene of a set ofone or more of the colorectal cancer marker genes: ANXA3, CLEC4D, IL2RB,LMNB1, PRRG4, TNFAIP6 and VNN1; a level of RNA encoded by the gene inblood of the test subject, thereby generating test data. In a secondstep, the method is effected by determining the probability that thetest data corresponds to positive control data representing levels ofRNA encoded by the gene in blood of human control subjects havingcolorectal cancer and not to negative control data representing levelsof RNA encoded by the gene in blood of human control subjects not havingcolorectal cancer. The probability that the test data corresponds to thepositive control data and not to the negative control data representsthe probability that the test subject has colorectal cancer as opposedto not having colorectal cancer.

Thus, according to an aspect of the invention, there is provided amethod of classifying a test subject as being more likely to havecolorectal cancer than to not have colorectal cancer. The method ofclassifying is effected by determining a level of RNA encoded by one ormore of the set of genes consisting of ANXA3, CLEC4D, IL2RB, LMNB1,PRRG4, TNFAIP6 and/or VNN1 in blood of the test subject, to therebygenerate test data and applying to the test data, and to negativecontrol data representing a level of RNA encoded by the gene in blood ofhuman control subjects not having colorectal cancer, a mathematicalformula for generating a value indicating, for ANXA3, CLEC4D, LMNB1,PRRG4, TNFAIP6 and VNN1, whether the level of RNA encoded by the gene inblood of the test subject is higher than the level of RNA encoded by thegene in blood of human control subjects not having colorectal cancer,and indicating, for IL2RB, whether the level of RNA encoded by the genein blood of the test subject is lower than the level of RNA encoded bythe gene in blood of human control subjects not having colorectalcancer. For ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1, andindication by the value that the level of RNA encoded by the gene inblood of the test subject is higher than the level of RNA encoded by thegene in blood of human control subjects not having colorectal cancerclassifies the test subject as more likely to have colorectal cancerthan to not have colorectal cancer; and where, for IL2RB, an indicationby the value that the level of RNA encoded by the gene in blood of thetest subject is lower than the level of RNA encoded by the gene in bloodof human control subjects not having colorectal cancer classifies thetest subject as more likely to have colorectal cancer than to not havecolorectal cancer.

Determining whether the level of RNA encoded by ANXA3, CLEC4D, LMNB1,PRRG4, TNFAIP6 or VNN1 in blood of the test subject is higher than thelevel of RNA encoded by the gene in blood of control subjects not havingcolorectal cancer may be effected by determining whether there is afold-change in the level between the test subject and the controlsubjects not having colorectal cancer which is higher than a minimumfold-change and/or which is within a range of fold-changes.

Determining whether the level of RNA encoded by IL2RB in blood of thetest subject is lower than the level of RNA encoded by the gene in bloodof control subjects not having colorectal cancer may be effected bydetermining whether there is a fold-change in the level between the testsubject and the control subjects not having colorectal cancer which islower than a maximum fold-change and/or which is within a range offold-changes.

Examples of suitable fold-changes and ranges of fold-changes forclassifying a test subject according to the invention are provided inExample 2, Example 3 and Example 6, below, and include the followingones.

For levels of RNA encoded by ANXA3, a suitable minimum fold-change isabout 1.6 fold, and a suitable range of fold-changes is about 1.6 toabout 11.5 fold, relative to an average level of RNA encoded by thehousekeeping gene in blood of subjects not having any colorectalpathology.

For levels of RNA encoded by CLEC4D, a suitable minimum fold-change iswhich is about 1.4 fold, and a suitable range of fold-changes is whichis about 1.4 to about 15.9 fold, relative to an average level of RNAencoded by the housekeeping gene in blood of subjects not having anycolorectal pathology.

For levels of RNA encoded by LMNB1, a suitable minimum fold-change isabout 1.3 fold, and a suitable range of fold-changes is about 1.3 toabout 7.0 fold, relative to an average level of RNA encoded by thehousekeeping gene in blood of subjects not having any colorectalpathology.

For levels of RNA encoded by PRRG4, a suitable minimum fold-change isabout 1.5 fold, and a suitable range of fold-changes is about 1.5 toabout 6.3 fold, relative to an average level of RNA encoded by thehousekeeping gene in blood of subjects not having any colorectalpathology.

For levels of RNA encoded by TNFAIP6, a suitable minimum fold-change isabout 1.4 fold, and a suitable range of fold-changes is about 1.45 toabout 16.8 fold, relative to an average level of RNA encoded by thehousekeeping gene in blood of subjects not having any colorectalpathology.

For levels of RNA encoded by VNN1, a suitable minimum fold-change isabout 1.5 fold, and a suitable range of fold-changes is about 1.45 toabout 23.6 fold, relative to an average level of RNA encoded by thehousekeeping gene in blood of subjects not having any colorectalpathology.

For levels of RNA encoded by IL2RB, a suitable maximum fold-change isabout 0.8 fold, and a suitable range of fold-changes is about 0.8 toabout 0.1 fold, relative to an average level of RNA encoded by thehousekeeping gene in blood of subjects not having any colorectalpathology.

For levels of RNA encoded by ANXA3 normalized to IL2RB, a suitableminimum fold-change is about 1.7 fold, and a suitable range offold-changes is about 1.7 to about 20.7 fold, relative to an averagelevel of RNA encoded by IL2RB in blood of subjects not having anycolorectal pathology.

For levels of RNA encoded by CLEC4D normalized to IL2RB, a suitableminimum fold-change is which is about 1.5 fold, and a suitable range offold-changes is which is about 1.5 to about 12.0 fold, relative to anaverage level of RNA encoded by IL2RB in blood of subjects not havingany colorectal pathology.

For levels of RNA encoded by LMNB1 normalized to IL2RB, a suitableminimum fold-change is about 1.5 fold, and a suitable range offold-changes is about 1.5 to about 10.6 fold, relative to an averagelevel of RNA encoded by IL2RB in blood of subjects not having anycolorectal pathology.

For levels of RNA encoded by PRRG4 normalized to IL2RB, a suitableminimum fold-change is about 1.3 fold, and a suitable range offold-changes is about 1.3 to about 13.1 fold, relative to an averagelevel of RNA encoded by IL2RB in blood of subjects not having anycolorectal pathology.

For levels of RNA encoded by TNFAIP6 normalized to IL2RB, a suitableminimum fold-change is about 1.5 fold, and a suitable range offold-changes is about 1.5 to about 16.4 fold, relative to an averagelevel of RNA encoded by IL2RB in blood of subjects not having anycolorectal pathology.

For levels of RNA encoded by VNN1 normalized to IL2RB, a suitableminimum fold-change is about 1.3 fold, and a suitable range offold-changes is about 1.3 to about 11.9 fold, relative to an averagelevel of RNA encoded by IL2RB in blood of subjects not having anycolorectal pathology.

As used herein, the term “about” refers to a variability of plus orminus 10 percent. Thus, a test subject of the invention is classified asbeing more likely to have colorectal cancer than to not have colorectalcancer if, for each marker gene of the particular set of marker genes ofthe invention used to practice the method of classifying of theinvention, the fold-change in level of RNA encoded by that gene in bloodof the test subject relative to blood of the control subjects not havingany colorectal cancer pathology classifies, according to the teachingsof the invention, the test subject as being more likely to havecolorectal cancer than to not have colorectal cancer.

Conversely, a test subject of the invention is classified as being morelikely to not have colorectal cancer than to have colorectal cancer if,for each marker gene of the particular set of marker genes of theinvention used to practice the method of classifying of the invention,the fold-change in level of RNA encoded by that gene in blood of thetest subject relative to blood of the control subjects not having anycolorectal cancer pathology does not classify, according to theteachings of the invention, the test subject as being more likely tohave colorectal cancer than to not have colorectal cancer.

In one aspect of the invention, the set of one or more colorectal cancermarker genes may consist of any one of the possible combinations of oneor more of ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1(indicated in Table 6, where each logistic regression model is based onone particular gene combination, and each gene of the combination isassigned a logistic regression coefficient value).

Sets of marker genes of the invention which consist of one or more ofANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1 which can be usedto practice the invention include: ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4,TNFAIP6, VNN1; ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, VNN1; ANXA3, CLEC4D,IL2RB, PRRG4; ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4; ANXA3, CLEC4D, IL2RB,PRRG4, VNN1; ANXA3, IL2RB, LMNB1, PRRG4, VNN1; ANXA3, CLEC4D, IL2RB,PRRG4, TNFAIP6; ANXA3, IL2RB, LMNB1, PRRG4, TNFAIP6; ANXA3, CLEC4D,IL2RB, LMNB1, PRRG4, TNFAIP6; ANXA3, CLEC4D, IL2RB, PRRG4, TNFAIP6,VNN1; ANXA3, IL2RB, LMNB1, PRRG4, TNFAIP6, VNN1; ANXA3, IL2RB, LMNB1,PRRG4; IL2RB, PRRG4, VNN1; ANXA3, IL2RB, PRRG4, VNN1; CLEC4D, IL2RB,PRRG4, VNN1; IL2RB, LMNB1, PRRG4, VNN1; CLEC4D, IL2RB, LMNB1, PRRG4,VNN1; ANXA3, IL2RB, PRRG4, TNFAIP6; IL2RB, PRRG4, TNFAIP6, VNN1; ANXA3,IL2RB, PRRG4, TNFAIP6, VNN1; CLEC4D, IL2RB, PRRG4, TNFAIP6, VNN1; IL2RB,LMNB1, PRRG4, TNFAIP6, VNN1; CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6, VNN1;IL2RB, PRRG4; ANXA3, IL2RB, PRRG4; CLEC4D, IL2RB, PRRG4; IL2RB, LMNB1,PRRG4; CLEC4D, IL2RB, LMNB1, PRRG4; IL2RB, PRRG4, TNFAIP6; CLEC4D,IL2RB, PRRG4, TNFAIP6; IL2RB, LMNB1, PRRG4, TNFAIP6; CLEC4D, IL2RB,LMNB1, PRRG4, TNFAIP6; ANXA3, IL2RB, VNN1; ANXA3, CLEC4D, IL2RB, VNN1;ANXA3, IL2RB, LMNB1, VNN1; ANXA3, CLEC4D, IL2RB, LMNB1, VNN1; ANXA3,CLEC4D, LMNB1, PRRG4, VNN1; ANXA3, IL2RB, TNFAIP6, VNN1; ANXA3, CLEC4D,IL2RB, TNFAIP6, VNN1; ANXA3, IL2RB, LMNB1, TNFAIP6, VNN1; ANXA3, CLEC4D,IL2RB, LMNB1, TNFAIP6, VNN1; ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6, VNN1;ANXA3, IL2RB; ANXA3, CLEC4D, IL2RB; ANXA3, IL2RB, LMNB1; ANXA3, CLEC4D,IL2RB, LMNB1; ANXA3, CLEC4D, LMNB1, PRRG4; CLEC4D, IL2RB, LMNB1, VNN1;ANXA3, IL2RB, TNFAIP6; ANXA3, CLEC4D, IL2RB, TNFAIP6; ANXA3, IL2RB,LMNB1, TNFAIP6; ANXA3, CLEC4D, IL2RB, LMNB1, TNFAIP6; ANXA3, CLEC4D,LMNB1, PRRG4, TNFAIP6; IL2RB, LMNB1, TNFAIP6, VNN1; CLEC4D, IL2RB,LMNB1, TNFAIP6, VNN1; IL2RB, LMNB1, VNN1; ANXA3, LMNB1, PRRG4, VNN1;ANXA3, LMNB1, PRRG4, TNFAIP6, VNN1; ANXA3, CLEC4D, PRRG4; ANXA3, LMNB1,PRRG4; CLEC4D, IL2RB, VNN1; ANXA3, CLEC4D, PRRG4, VNN1; IL2RB, LMNB1,TNFAIP6; CLEC4D, IL2RB, LMNB1, TNFAIP6; ANXA3, CLEC4D, PRRG4, TNFAIP6;ANXA3, LMNB1, PRRG4, TNFAIP6; IL2RB, TNFAIP6, VNN1; CLEC4D, IL2RB,TNFAIP6, VNN1; ANXA3, CLEC4D, PRRG4, TNFAIP6, VNN1; IL2RB, LMNB1;CLEC4D, IL2RB, LMNB1; IL2RB, VNN1; ANXA3, CLEC4D, LMNB1, VNN1; ANXA3,CLEC4D, LMNB1, TNFAIP6, VNN1; ANXA3, CLEC4D, LMNB1; ANXA3, PRRG4; ANXA3,CLEC4D, VNN1; ANXA3, LMNB1, VNN1; ANXA3, PRRG4, VNN1; ANXA3, CLEC4D,LMNB1, TNFAIP6; ANXA3, PRRG4, TNFAIP6; ANXA3, CLEC4D, TNFAIP6, VNN1;ANXA3, LMNB1, TNFAIP6, VNN1; ANXA3, PRRG4, TNFAIP6, VNN1; ANXA3; ANXA3,CLEC4D; ANXA3, LMNB1; ANXA3, VNN1; ANXA3, TNFAIP6; ANXA3, CLEC4D,TNFAIP6; IL2RB, TNFAIP6; CLEC4D, IL2RB, TNFAIP6; ANXA3, LMNB1, TNFAIP6;ANXA3, TNFAIP6, VNN1; CLEC4D, IL2RB; PRRG4, VNN1; CLEC4D, PRRG4, VNN1;LMNB1, PRRG4, VNN1; CLEC4D, LMNB1, PRRG4, VNN1; PRRG4, TNFAIP6, VNN1;CLEC4D, PRRG4, TNFAIP6, VNN1; LMNB1, PRRG4, TNFAIP6, VNN1; CLEC4D,LMNB1, PRRG4, TNFAIP6, VNN1; PRRG4; CLEC4D, PRRG4; LMNB1, PRRG4; CLEC4D,LMNB1, PRRG4; PRRG4, TNFAIP6; CLEC4D, PRRG4, TNFAIP6; LMNB1, PRRG4,TNFAIP6; CLEC4D, LMNB1, PRRG4, TNFAIP6; LMNB1, TNFAIP6, VNN1; CLEC4D,VNN1; LMNB1, VNN1; CLEC4D, LMNB1, VNN1; LMNB1, TNFAIP6; LMNB1, TNFAIP6;TNFAIP6, VNN1; CLEC4D, TNFAIP6, VNN1; CLEC4D, LMNB1, TNFAIP6, VNN1;LMNB1; CLEC4D, LMNB1; VNN1; CLEC4D, TNFAIP6; TNFAIP6; CLEC4D; and IL2RB.

According to the aspect of the invention where the set of one or morecolorectal cancer marker genes consists of any one of the 127 possiblecombinations of ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1,the level of RNA encoded by a gene of the invention in blood of asubject of the invention may be determined as a ratio to a level of RNAencoded by a housekeeping gene in blood of the subject. It will beappreciated that such measurement of a level or RNA encoded by a generelative to that of a housekeeping gene within individual samples can beused to control for sample to sample variability.

The housekeeping gene may be any one of various genes expressed in bloodknown to the ordinarily skilled artisan. In one aspect of the method,the housekeeping gene is ACTB. Alternately, the housekeeping gene mayencode 18S rRNA.

Nucleotide sequences of target genes of the invention (ACTB, ANXA3,CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1) are described in FigureslA-H and in Table 1, below.

In another aspect of the invention, the set of one or more colorectalcancer marker genes may consist of any one of the possible combinationsof one or more of ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1(indicated in Table 5, where each logistic regression model is based onone particular gene combination, and each gene of the combination isassigned a logistic regression coefficient value).

The possible combinations of one or more of ANXA3, CLEC4D, LMNB1, PRRG4,TNFAIP6 and VNN1 which can be used to practice the invention include:ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1; ANXA3, LMNB1, PRRG4,TNFAIP6 and VNN1; ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6; ANXA3, CLEC4D,LMNB1, PRRG4, TNFAIP6 and VNN1; ANXA3, PRRG4, TNFAIP6 and VNN1; CLEC4D,LMNB1, PRRG4, TNFAIP6 and VNN1; ANXA3, PRRG4 and TNFAIP6; CLEC4D, LMNB1,PRRG4 and TNFAIP6; ANXA3, CLEC4D, PRRG4, TNFAIP6 and VNN1; ANXA3,CLEC4D, PRRG4 and TNFAIP6; ANXA3, LMNB1, PRRG4 and VNN1; ANXA3, LMNB1,PRRG4 and TNFAIP6; CLEC4D, LMNB1, PRRG4 and VNN1; ANXA3, CLEC4D, LMNB1,PRRG4; ANXA3, CLEC4D, PRRG4 and VNN1; LMNB1, PRRG4 and VNN1; LMNB1,PRRG4, TNFAIP6 and VNN1; LMNB1, PRRG4 and TNFAIP6; ANXA3, CLEC4D andPRRG4; ANXA3, LMNB1 and PRRG4; ANXA3 and PRRG4; ANXA3, PRRG4 and VNN1;CLEC4D, LMNB1 and PRRG4; LMNB1 and PRRG4; CLEC4D, PRRG4, TNFAIP6 andVNN1; CLEC4D, PRRG4 and TNFAIP6; CLEC4D, PRRG4 and VNN1; CLEC4D andPRRG4; PRRG4, TNFAIP6 and VNN1; PRRG4 and VNN1; PRRG4 and TNFAIP6;PRRG4; TNFAIP6 and VNN1; VNN1; ANXA3, TNFAIP6 and VNN1; ANXA3, LMNB1,TNFAIP6 and VNN1; LMNB1, TNFAIP6 and VNN1; CLEC4D, TNFAIP6 and VNN1;ANXA3, CLEC4D, TNFAIP6 and VNN1; ANXA3, CLEC4D, LMNB1, TNFAIP6 and VNN1;CLEC4D, LMNB1, TNFAIP6 and VNN1; ANXA3 and VNN1; ANXA3, CLEC4D, LMNB1and TNFAIP6; CLEC4D, LMNB1 and TNFAIP6; CLEC4D and VNN1; LMNB1 and VNN1;ANXA3, CLEC4D and VNN1; ANXA3, LMNB1 and VNN1; ANXA3, LMNB1 and TNFAIP6;LMNB1 and TNFAIP6; CLEC4D, LMNB1 and VNN1; ANXA3, CLEC4D, LMNB1 andVNN1; ANXA3, CLEC4D and TNFAIP6; CLEC4D and TNFAIP6; CLEC4D, LMNB1;ANXA3, CLEC4D and LMNB1; LMNB1; ANXA3 and TNFAIP6; ANXA3 and LMNB1;TNFAIP6; ANXA3 and CLEC4D; CLEC4D; and ANXA3.

According to the aspect of the invention where the set of one or morecolorectal cancer marker genes consists of any one of the 63 possiblecombinations of ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1, the levelof RNA encoded by a gene of the invention in blood of a subject of theinvention may be determined as a ratio to a level of RNA encoded byIL2RB in blood of the subject.

It will be appreciated that data representing levels of RNA encoded by aset of genes of the invention may be combined with data representinglevels of gene products of other genes which are differently expressedin blood in subjects having colorectal cancer relative to subjects nothaving any colorectal pathology so as to determine a probability that atest subject has colorectal cancer versus not having any colorectalpathology.

In another aspect, the method further comprises determining levels ofRNA encoded by the gene in blood of a population of control humansubjects having colorectal cancer, and/or in blood of a population ofhuman control subjects not having colorectal cancer, to thereby providethe positive control data and/or the negative control data,respectively. Alternately, it is envisaged that the level of RNA encodedby a gene of the invention in control subjects of the invention could beprovided by prior art data corresponding to control data of theinvention.

The method of the invention may be practiced using any one of varioustypes of control subjects.

In an aspect of the method of the invention, the control subjects nothaving colon cancer are subjects having been diagnosed as not having anycolorectal pathology as a result of colonoscopic examination. As isdescribed in the Examples section which follows, the method of theinvention may be practiced using subjects not having any colorectalpathology as the control subjects not having colorectal cancer.

In an aspect of the method of the invention, the control subjects havingcolorectal cancer are subjects having been diagnosed as havingcolorectal cancer as a result of colonoscopic examination. As isdescribed in the Examples section which follows, the method of theinvention may be practiced using subjects diagnosed as not having anycolorectal pathology as the control subjects not having colorectalcancer.

The method of the invention may furthermore be practiced using any oneof various numbers of control subjects. One of ordinary skill in the artwill possess the necessary expertise to select a sufficient number ofcontrol subjects so as to obtain control data having a desiredstatistical significance for practicing the method of the invention witha desired level of reliability.

For example, the method of the invention can be practiced using 10 ormore, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 ormore, 80 or more, 90 or more, 100 or more, 10 or more, 20 or more, 30 ormore, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 ormore, 100 or more, 110 or more, 120 or more, 130 or more, 140 or more,150 or more, 160 or more, 170 or more, 180 or more, 190 or more, or 200or more of control subjects having colorectal cancer and/or of controlsubjects not having colorectal cancer.

In one aspect of the invention, the level of RNA encoded by a gene ofthe invention in blood of the test subject and the levels of RNA encodedby the gene in blood of the control subjects are determined via the samemethod. As is described in the Examples section, below, the method canbe practiced where the level of RNA encoded by a gene of the inventionin blood of the test subject and the levels of RNA encoded by the genein blood of the control subjects are determined via the same method.Alternately, it is envisaged that the level of a gene of the inventionin blood of a test subject of the invention and in blood of controlsubjects of the invention could be determined using different methods.It will be appreciated that use of the same method to determine thelevels of RNA encoded by a gene of the invention in a test subject andin control subjects of the invention can be used to avoidmethod-to-method calibration to minimize any variability which mightarise from use of different methods.

In one aspect of the method, determining of the level of RNA encoded bya gene of the invention in blood of a subject of the invention iseffected by determining the level of RNA encoded by the gene in a bloodsample isolated from the subject. Alternately, it is envisaged thatdetermining of the level of RNA encoded by the gene in blood of asubject of the invention could be effected by determining the level ofRNA encoded by the gene in an in-vivo sample using a suitable method forsuch a purpose.

In one aspect of the method, the level of RNA encoded by a gene of theinvention in blood of a subject of the invention is determined in asample of RNA isolated from blood of the subject. Alternately, it isenvisaged that the level of RNA of a gene of the invention in blood of asubject of the invention could be determined in a sample which includesRNA of blood of the subject but from which RNA has not been isolatedtherefrom, using a suitable method for such a purpose.

Any one of various methods routinely employed in the art for isolatingRNA from blood may be used to isolate RNA from blood of a subject of theinvention, so as to enable practicing of the method of the invention.

In one aspect of the method, the level of RNA encoded by a gene of theinvention in blood of a subject of the invention is determined in RNA ofa sample of whole blood. Any one of various methods routinely employedin the art for isolating RNA from whole blood may be employed forpracticing the method.

Alternately, it is envisaged that the level of RNA encoded by a gene ofthe invention in blood of a subject of the invention could be determinedin RNA of a sample of fraction of blood which expresses the genesufficiently specifically so as to enable the method. Examples of suchblood fractions include preparations of isolated types of leukocytes,preparations of isolated peripheral blood mononuclear cells,preparations of isolated granulocytes, preparations of isolated wholeleukocytes, preparations of isolated specific types of leukocytes,plasma-depleted blood, preparations of isolated lymphocytes, and theplasma fraction of blood.

In one aspect of the method, isolation of RNA from whole blood of asubject of the invention is effected by using a PAXgene Blood RNA Tube(obtainable from PreAnalytiX) in accordance with the instructions of thePAXgene Blood RNA Kit protocol. As is described in the Examples sectionbelow, the method of the invention may be practiced by determining alevel of a gene of the invention in RNA isolated from blood from testand control subjects of the invention using PAXgene Blood RNA Tubes.

Determining of a level of RNA encoded by a gene of the invention in asample of the invention may be effected in any one of various waysroutinely practiced in the art.

For example, the level of RNA encoded by a gene of the invention in asample of the invention may be determined via any one of various methodsbased on quantitative polynucleotide amplification which are routinelyemployed in the art for determining a level of RNA encoded by a gene ina sample.

Alternately, the level of RNA encoded by a gene of the invention may bedetermined via any one of various methods based on quantitativepolynucleotide hybridization to an immobilized probe which are routinelyemployed in the art for determining a level of RNA encoded by a gene ina sample.

In one aspect of the method of the invention, the method based onquantitative polynucleotide amplification used to determine the level ofRNA encoded by a gene of the invention is quantitative reversetranscriptase-polymerase chain reaction (PCR) analysis. Any one ofvarious types of quantitative reverse transcriptase-PCR analysesroutinely employed in the art to determine the level of RNA encoded by agene in a sample may be used to practice the invention. For example, anyone of various sets of primers may be used to perform quantitativereverse transcriptase-PCR analysis so as to practice the method of theinvention.

In one aspect of the method of the invention, the quantitative reversetranscriptase-PCR analysis used to determine the level of RNA encoded bya gene of the invention is quantitative real-time PCR analysis of DNAcomplementary to RNA encoded by the gene using a labeled probe capableof specifically binding amplification product of DNA complementary toRNA encoded by the gene. For example, quantitative real-time PCRanalysis may be performed using a labeled probe which comprises apolynucleotide capable of selectively hybridizing with a sense orantisense strand of amplification product of DNA complementary to RNAencoded by the gene. Labeled probes comprising a polynucleotide havingany one of various nucleic acid sequences capable of specificallyhybridizing with amplification product of DNA complementary to RNAencoded by the gene may be used to practice the method of the invention.

Quantitative real-time PCR analysis of a level of RNA encoded by a geneof the invention may be performed in any one of various ways routinelyemployed in the art.

In one aspect of the method of the invention, quantitative real-time PCRanalysis is performed by analyzing complementary DNA prepared from RNAof blood a subject of the invention, using the QuantiTect™ Probe RT-PCRsystem (Qiagen, Valencia, Calif.; Product Number 204345), a TaqMan duallabelled probe, and a Real-Time PCR System 7500 instrument (AppliedBiosystems). As is described in the Examples section which follows, suchquantitative real-time PCR analysis may be used to practice the methodof the invention.

As specified above, the level of RNA encoded by a gene of the inventionmay be determined via a method based on quantitative polynucleotidehybridization to an immobilized probe.

In one aspect, determining of the level of RNA encoded by a gene of theinvention via a method based on quantitative polynucleotidehybridization is effected using a microarray, such as an AffymetrixU133Plus 2.0 GeneChip oligonucleotide array (Affymetrix; Santa Clara,Calif.).

As specified above, the level of RNA encoded by a gene of the inventionin a sample of the invention may be determined via quantitative reversetranscriptase-PCR analysis using any one of various sets of primers andlabeled probes to amplify and quantitate DNA complementary to RNAencoded by a marker gene of the invention produced during such analysis.Examples of suitable primers for use in quantitative reversetranscriptase-PCR analysis of the level of RNA encoded by a target geneof the invention are listed in Table 19. This table further listsexamples of suitable polynucleotides comprised in labeled probes forpracticing quantitative real-time PCR analysis according to the methodof the invention.

TABLE 19PCR primers and matching polynucleotides of labeled probes for quantitative PCR analysis.Gene encoding Primer/ amplified AssayNucleic acid sequences of PCR primers and matching probe Amplicon cDNAreagent polynucleotides comprised in labeled probes position size (bp)ACTB 5′ 5′-CACCACACCTTCTACAATGAGCTG-3′ (SEQ ID NO: 1) 259 158 primer5′-ACAGCCTGGATAGCAACGTACA-3′ (SEQ ID NO: 2) 416 3′5′-AACCGCGAGAAGATGACCCAGATCAT-3′ (SEQ ID NO: 3) 343 primer probe 5′5′-ACCTTCTACAATGAGCTGCG-3′ (SEQ ID NO: 4) 337 114 primer5′-GGTCTCAAACATGATCTGGGTC-3′ (SEQ ID NO: 5) 450 3′5′-AAGGCCAACCGCGAGAAGAT-3′ (SEQ ID NO: 6) 409 primer probe 5′5′-CACCCAGCACAATGAAGATC-3′ (SEQ ID NO: 7) 1034 119 primer5′-CTGCTTGCTGATCCACATCT-3′ (SEQ ID NO: 8) 1152 3′5′-ATCATTGCTCCTCCTGAGCG-3′ (SEQ ID NO: 9) 1057 primer probe ANXA3 5′5′-GAAACATCTGGTGACTTCCG-3′ (SEQ ID NO: 10) 748 103 primer5′-TCTGGGCATCTTGTTTGG-3 ′ (SEQ ID NO: 11) 850 3′5′-TTGACTTTGGCAGATGGCAGA-3′ (SEQ ID NO: 12) 778 primer probe 5′5′-GGAACAAACGAAGATGCCTTG-3′ (SEQ ID NO: 13) 628 137 primer5′-AAGTCACCAGATGTTTCGGA-3′ (SEQ ID NO: 14) 764 3′5′-ATCTTAACTACCAGGACAAGCAGGCA-3′ (SEQ ID NO: 15) 655 primer probe 5′5′-CTACCAGGACAAGCAGGCAA-3′ (SEQ ID NO: 16) 662 138 primer5′-TTCTGCCATCTGCCAAAGT-3′ (SEQ ID NO: 17) 799 3′5′-TCCGAAACATCTGGTGACTTCC-3′ (SEQ ID NO: 18) 745 primer probe CLEC4D 5′5′-CCATTTAACCCACGCAGAG-3′ (SEQ ID NO: 19) 673 101 primer5′-CAGGCCCATTTATCTTGGTT-3′ (SEQ ID NO: 20) 773 3′5′-CTGGCATAAGAATGAACCCGACA-3′ (SEQ ID NO: 21) 696 primer probe 5′5′-TCCGAAACATCTGGTGACTTCC-3′ (SEQ ID NO: 22) 406 118 primer5′-TCCTTTCACTCTCAGCCCAC-3′ (SEQ ID NO: 23) 523 3′5′-ATGACCATCAGCACGGAAGC-3′ (SEQ ID NO: 24) 550 primer probe 5′5′-GGGCTGAGAGTGAAAGGAAC-3′ (SEQ ID NO: 25) 506 149 primer5′-CCACTGACCTTTGGCATTC-3′ (SEQ ID NO: 26) 654 3′5′-ATGACCATCAGCACGGAAGC-3′ (SEQ ID NO: 27) 550 primer probe IL2RB 5′5′-AAATCTCCCAAGCCTCCCA-3′ (SEQ ID NO: 28) 588 127 primer5′-AGGCAGATCCATTCCTGCT-3′ (SEQ ID NO: 29) 714 3′5′-TTGAAAGACACCTGGAGTTCG-3 ′ (SEQ ID NO: 30) 612 primer probe 5′5′-GACCCACAGATGCAACATAAG-3′ (SEQ ID NO: 31) 562 137 primer5′-GCTTCTGCTTGAGAGTCAGC-3′ (SEQ ID NO: 32) 698 3′5′-AAATCTCCCAAGCCTCCCAC-3′ (SEQ ID NO: 33) 588 primer probe 5′5′-TGGAGACCCACAGATGCAA-3′ (SEQ ID NO: 34) 558 141 primer5′-GCTTCTGCTTGAGAGTCAGC-3′ (SEQ ID NO: 35) 698 3′5′-AAATCTCCCAAGCCTCCCAC-3′ (SEQ ID NO: 36) 588 primer probe LMNB1 5′5′-GGAGTGGTTGTTGAGGAAGAA-3′ (SEQ ID NO: 37) 2051 151 primer5′-CTGAGAAGGCTCTGCACTGTA-3 ′ (SEQ ID NO: 38) 2201 3′5′-AACCCCAAGAGCATCCAATAG-3′ (SEQ ID NO: 39) 2089 primer probe 5′5′-CTGGCGAAGATGTGAAGGT-3′ (SEQ ID NO: 40) 1935 135 primer5′-CTTCCTCAACAACCACTCCA-3′ (SEQ ID NO: 41) 2069 3′5′-AATTCTCAGGGAGAGGAGGTTG-3′ (SEQ ID NO: 42) 1964 primer probe 5′5′-AGGCGAAGAAGAGAGGTTGAAG-3′ (SEQ ID NO: 43) 1513 103 primer5′-CCGCTTTCCTCTAGTTGTACG-3′ (SEQ ID NO: 44) 1615 3′5′-TGTCTCCAAGCCCTTCTTCC-3′ (SEQ ID NO: 45) 1536 primer probe PRRG4 5′5′-ATGCGGGAGAAGAAGTGTTTAC-3′ (SEQ ID NO: 46) 341 153 primer5′-CTCTGGCTTCCTCATAATTGC-3′ (SEQ ID NO: 47) 493 3′5′-CTCTTCACTCCCGGCAACCTAGAA-3′ (SEQ ID NO: 48) 427 primer probe 5′5′-TGCTGCTGGAGTATTTTTGG-3′ (SEQ ID NO: 49) 618 130 primer5′-AATGATGGAGGGAGTGTGC-3′ (SEQ ID NO: 50) 747 3′5′-AACATCCATGCTCTTCAGCC-3′ (SEQ ID NO: 51) 693 primer probe 5′5′-ACTCCCGGCAACCTAGAAAG-3′ (SEQ ID NO: 52) 433 176 primer5′-GTCAGAAGGCCCATAACATCTA-3′ (SEQ ID NO: 53) 608 3′5′-AACGATTGCATTTTGGCAGG-3′ (SEQ ID NO: 54) 517 primer probe TNFAIP6 5′5′-GCCTATTGCTACAACCCACA-3′ (SEQ ID NO: 55) 448 84 primer5′-TGGGAAGCCTGGAGATTTA-3′ (SEQ ID NO: 56) 531 3′5′-AAGGAGTGTGGTGGCGTCTTTAC-3′ (SEQ ID NO: 57) 472 primer probe 5′5′-CAGGTTGCTTGGCTGATTATG-3 ′ (SEQ ID NO: 58) 632 172 primer5′-TTGATTTGGAAACCTCCAGC-3′ (SEQ ID NO: 59) 803 3′5′-TGGCTTTGTGGGAAGATACTGTGG-3′ (SEQ ID NO: 60) 684 primer probe 5′5′-CATTAGACTCAAGTATGGTCAGCG-3′ (SEQ ID NO: 61) 567 142 primer5′-TCCACAGTATCTTCCCACAAAG-3′ (SEQ ID NO: 62) 708 3′5′-CAGGTTGCTTGGCTGATTATGT-3′ (SEQ ID NO: 63) 632 primer probe VNN1 5′5′-TGACAGGAAGTGGCATCTAT-3′ (SEQ ID NO: 64) 835 147 primer5′-TACTGCTGGCATAGGAAGTC-3′ (SEQ ID NO: 65) 981 3′5′-AGAAGAGGGAAAACTCCTCCTCTCG-3′ (SEQ ID NO: 66) 896 primer probe 5′5′-CTGGAGAATTTCAGGTGTCA-3′ (SEQ ID NO: 67) 1360 111 primer5′-ATGCCCAGTCCTTCTCATAC-3′ (SEQ ID NO: 68) 1470 3′5′-ACTGACGGACGCTTGTTTAGTCTGA-3′ (SEQ ID NO: 69) 1380 primer probe 5′5′-GTATTCCCAACAGCTTGGAT-3′ (SEQ ID NO: 70) 711 144 primer5′-ATAGATGCCACTTCCTGTCA-3′ (SEQ ID NO: 71) 854 3′5′-CATGAGGGTCAATTTCCTTGCATC-3′ (SEQ ID NO: 72) 785 primer probe

Determining the level of RNA encoded by the marker gene of the inventionas a ratio to a housekeeping gene may be effected in any one of variousways routinely employed in the art for determining a ratio of a level ofRNA encoded by one gene to a level of RNA encoded by a housekeepinggene, such as ACTB.

In one aspect of the method, determining the level of RNA encoded by thegene of the invention as a ratio to the housekeeping gene is effectedvia duplex quantitative reverse transcriptase-PCR analysis of RNAencoded by the gene and of RNA encoded by the housekeeping gene in asample of the invention. Such “duplex quantitative reverse transcriptasePCR analysis” refers to quantitative reverse transcriptase-PCR analysiswhere DNA complementary to RNA encoded by the gene of the invention andDNA complementary to RNA encoded by the housekeeping gene areco-amplified in the same sample/reaction mixture.

DNA complementary to RNA encoded by the housekeeping gene may beamplified via quantitative reverse transcriptase-PCR analysis using anyone of various suitable primers.

In one aspect, the primers may be selected so as to include a primerhaving a nucleotide sequence which is complementary to a region of atarget cDNA template, where the region spans a splice junction joining apair of exons. It will be appreciated that such a primer can be used tofacilitate amplification of DNA complementary to messenger RNA, i.e.mature spliced RNA.

In one aspect of the method, where the housekeeping gene is ACTB, theprimers used to amplify DNA complementary to RNA encoded by thehousekeeping gene may include a primer having a nucleotide sequenceidentified as SEQ ID NO: 1, a primer having a nucleotide sequenceidentified as SEQ ID NO: 2, or both primers.

In another aspect of the method, the level of RNA encoded by thehousekeeping gene in blood of the test subject is determined viaquantitative reverse transcriptase-PCR analysis, using a labeled probewhich comprises a polynucleotide capable of hybridizing to a sense orantisense strand of the amplification product of the DNA complementaryto RNA encoded by the housekeeping gene.

In one aspect of the method where the housekeeping gene is ACTB andwhere the level of RNA encoded by the housekeeping gene in blood of thetest subject is determined via quantitative reverse transcriptase-PCRanalysis using a primer having a nucleotide sequence identified as SEQID NO: 1, and a primer having a nucleotide sequence identified as SEQ IDNO: 2, and a labeled probe, the probe comprises a polynucleotide havinga nucleic acid sequence identified as SEQ ID NO: 3.

As is demonstrated in Example 2 of the Examples section which follows,the method of the invention can be practiced by determining the level ofRNA encoded by any one of the marker genes ANXA3, CLEC4D, IL2RB, LMNB1,PRRG4, TNFAIP6 and VNN1 as a ratio to a level of RNA encoded by ACTB inblood of a subject of the invention, where the level is determined viaduplex quantitative reverse transcriptase-PCR analysis using a primerhaving a nucleotide sequence identified as SEQ ID NO: 1, a primer havinga nucleotide sequence identified as SEQ ID NO: 2, and a labeled probewhich comprises a polynucleotide having a nucleic acid sequenceidentified as SEQ ID NO: 3.

Determining the level of RNA encoded by ANXA3, CLEC4D, LMNB1, PRRG4,TNFAIP6 or VNN1 as a ratio to IL2RB may be effected in any one ofvarious ways.

In one aspect of the method, determining the level of ANXA3, CLEC4D,LMNB1, PRRG4, TNFAIP6 or VNN1 as a ratio to a level of RNA encoded byIL2RB in a sample of the invention is effected via duplex quantitativereverse transcriptase-PCR analysis of RNA encoded by ANXA3, CLEC4D,LMNB1, PRRG4, TNFAIP6 or VNN1 and of RNA encoded by IL2RB in the sample.Such “duplex quantitative reverse transcriptase PCR analysis” refers toquantitative reverse transcriptase-PCR analysis where DNA complementaryto RNA encoded by ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 or VNN1 and DNAcomplementary to RNA encoded by IL2RB are co-amplified in the samesample/reaction mixture.

As described above, following the step of obtaining the test data, themethod of the invention comprises the step of determining theprobability that the test data corresponds to the positive control dataand not to the negative control data.

It will be appreciated that the probability that the test subject doesnot have any colorectal pathology as opposed to having colorectal cancercan be readily determined from the probability that the test subject hascolorectal cancer as opposed to not having colorectal cancer. Forexample, when expressing the probability that the test subject hascolorectal cancer as a percentage probability, the probability that thetest subject does not have any colorectal pathology as opposed to havingcolorectal cancer corresponds to 100 percent minus the probability thatthe test subject does not have any colorectal pathology as opposed tohaving colorectal cancer.

Determining the probability that the test data corresponds to thepositive control data and not to the negative control data may beeffected in any one of various ways known to the ordinarily skilledartisan for determining the probability that a gene expression profileof a test subject corresponds to a gene expression profile of ofsubjects having a pathology and not to a gene expression profile ofsubjects not having the pathology, where the gene expression profiles ofthe subjects having the pathology and the subjects not having thepathology are significantly different.

In one aspect of the method, determining the probability that the testdata corresponds to the positive control data and not to the negativecontrol data is effected by applying to the test data a mathematicalmodel derived from the positive control data and from the negativecontrol data.

Various suitable mathematical models which are well known in the art ofmedical diagnosis using disease markers may be employed to classify atest subject as more likely to have colorectal cancer than to not havecolorectal cancer, to determine a probability that a test subject islikely to have colorectal cancer as opposed to not having colorectalcancer, or to diagnose a test subject as having colorectal canceraccording to the teachings of the invention. Generally thesemathematical models can be unsupervised methods performing a clusteringwhilst supervised methods are more suited to classification of datasets.(refer, for example, to: Dreiseitl S, Ohno-Machado L. Logisticregression and artificial neural network classification models: amethodology review. J Biomed Inform. 2002 October-December;35(5-6):352-9; Pepe M S. The Statistical Evaluation of Medical Tests forClassification and Prediction. Oxford, England: Oxford University Press;2003; Dupont W D. Statistical Modeling for Biomedical Researchers.Cambridge, England: Cambridge University Press; 2002; Pampel F C.Logistic regression: A Primer. Publication #07-132, Sage Publications:Thousand Oaks, Calif. 2000; King E N, Ryan T P. A preliminaryinvestigation of maximum likelihood logistic regression versus exactlogistic regression. Am Statistician 2002; 56:163-170; Metz C E. Basicprinciples of ROC analysis. Semin Nucl Med 1978; 8:283-98; Swets J A.Measuring the accuracy of diagnostic systems. Science 1988; 240:1285-93;Zweig M H, Campbell G. Receiver-operating characteristic (ROC) plots: afundamental evaluation tool in clinical medicine. Clin Chem 1993;39:561-77; Witten I H, Frank Eibe. Data Mining: Practical MachineLearning Tools and Techniques (second edition). Morgan Kaufman 2005;Deutsch J M. Evolutionary algorithms for finding optimal gene sets inmicroarray prediction. Bioinformatics 2003; 19:45-52; Niels Landwehr,Mark Hall and Eibe Frank (2003) Logistic Model Trees. pp 241-252 inMachine Learning: ECML 2003: 14th European Conference on MachineLearning, Cavtat-Dubrovnik, Croatia, September 22-26, 2003, ProceedingsPublisher: Springer-Verlag GmbH, ISSN: 0302-9743). Examples of suchmathematical models, related to learning machine, include: RandomForests methods, logistic regression methods, neural network methods,k-means methods, principal component analysis methods, nearest neighbourclassifier analysis methods, linear discriminant analysis, methods,quadratic discriminant analysis methods, support vector machine methods,decision tree methods, genetic algorithm methods, classifieroptimization using bagging methods, classifier optimization usingboosting methods, classifier optimization using the Random Subspacemethods, projection pursuit methods, genetic programming and weightedvoting methods.

In one aspect of the invention, the model used is a logistic regressionmodel. As is described in the Examples section below, logisticregression models can be used according to the method of the inventionto determine the probability a test subject of the invention hascolorectal cancer as opposed to not having any colorectal pathology.Logistic regression models may also be referred to in the art as“logistic models”, and “logit models”.

Any one of various particular cases of logistic regression models may beused, for any given set of genes of the invention, for determining theprobability that the test data corresponds to the positive control dataand not to the negative control data.

In one aspect of the method, determining the probability that the testdata corresponds to the positive control data and not to the negativecontrol data is effected by using one or more of the logistic regressionmodels disclosed in Example 2, Example 3 and Example 6.

It will be appreciated that a computer may be used for determining theprobability that the test subject has colorectal cancer using amathematical model, according to the method of the invention.

One of skill in the art will know of suitable mathematical formulas forgenerating a value indicating whether the level of RNA encoded by thegene in blood of the test subject is higher or lower than the level ofRNA encoded by the gene in blood of human control subjects not havingcolorectal cancer.

For example, a suitable formula, is one which generates a valuerepresenting the ratio of the level of RNA encoded by the gene in bloodof the test subject to the level of RNA encoded by the gene in blood ofhuman control subjects not having colorectal cancer. A ratio of greaterthan 1 indicates that the level of RNA encoded by the gene in blood ofthe test subject is higher than the level of RNA encoded by the gene inblood of human control subjects not having colorectal cancer, and aratio of less than 1 indicates that the level of RNA encoded by the genein blood of the test subject is lower than the level of RNA encoded bythe gene in blood of human control subjects not having colorectalcancer. A formula for generating such a ratio value may have the form:

Value=[level of RNA encoded by the gene in blood of the testsubject]/[level of RNA encoded by the gene in blood of human controlsubjects not having colorectal cancer]

Alternately, a suitable formula is one which subtracts the level of RNAencoded by the gene in blood of human control subjects not havingcolorectal cancer from the level of RNA encoded by the gene in blood ofthe test subject, to generate a value representing the differencebetween the level of RNA encoded by the gene in blood of the testsubject from the level of RNA encoded by the gene in blood of humancontrol subjects not having colorectal cancer. A difference having apositive value indicates that the level of RNA encoded by the gene inblood of the test subject is higher than the level of RNA encoded by thegene in blood of human control subjects not having colorectal cancer,and a difference having a negative value indicates that the level of RNAencoded by the gene in blood of the test subject is lower than the levelof RNA encoded by the gene in blood of human control subjects not havingcolorectal cancer. A formula for generating such a difference value mayhave the form:

Value=[level of RNA encoded by the gene in blood of the testsubject]−[level of RNA encoded by the gene in blood of human controlsubjects not having colorectal cancer]

Thus, according to another aspect of the invention there is provided acomputer-based method of determining the probability that a test subjecthas colorectal cancer as opposed to not having colorectal cancer. Themethod is effected by causing a computer to apply to the test data amathematical model according to the invention, and to output theprobability, to thereby enable a determination of the probability thatthe test subject has colorectal cancer as opposed to not havingcolorectal cancer.

Application of computers for determining a probability that a testsubject has a disease as opposed to not having the disease, so as toenable the method of the invention, is routinely practiced in the artusing computer systems, and optionally computer-readable media,routinely used in the art.

Thus, according to a further aspect of the invention there is provided acomputer system for providing the probability that the test subject hascolorectal cancer as opposed to not having colorectal cancer. Thecomputer system comprises a processor; and a memory configured withinstructions that cause the processor to provide a user with theprobability, where the instructions comprise applying a mathematicalmodel of the invention to test data of the invention, to therebydetermine the probability that the test subject has colorectal cancer asopposed to not having colorectal cancer.

The instructions may be provided to the computer in any one of variousways routinely employed in the art. In one aspect, the instructions areprovided to the computer using a computer-readable medium.

Thus, according to yet another aspect of the invention there is provideda computer-readable medium having instructions stored thereon that areoperable when executed by a computer for applying a mathematical modelof the invention to test data of the invention from, thereby determinethe probability that a test subject has colorectal cancer as opposed tonot having colorectal cancer.

As described above, following the step of obtaining the test data, themethod of classifying of the invention comprises the step of comparingtest data representing a level of RNA encoded by a marker gene of theinvention to negative control data representing a level of RNA encodedby the gene in subjects not having any colorectal pathology, anddetermining the fold-change between the levels.

It will be appreciated that a computer may be used for comparing testdata representing a level of RNA encoded by a marker gene of theinvention to negative control data representing a level of RNA encodedby the gene in subjects not having any colorectal pathology, anddetermining the fold-change between the levels, according to methods ofthe invention. Thus, according to another aspect of the invention thereis provided a computer-based method of classifying a human test subjectas more likely to have colorectal cancer than to not have colorectalcancer. The method is effected by using a computer to apply to test datafrom a test subject according to the invention, and to negative controldata representing a level of RNA encoded by the gene in blood of humancontrol subjects not having colorectal cancer, a mathematical formulafor generating a value indicating whether the level of RNA encoded bythe gene in blood of the test subject is higher, for ANXA3, CLEC4D,LMNB1, PRRG4, TNFAIP6 and VNN1, or lower, for IL2RB, than the level ofRNA encoded by the gene in blood of human control subjects not havingcolorectal cancer. For ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1, anindication by the value that the level of RNA encoded by the gene inblood of the test subject is higher than the level of RNA encoded by thegene in blood of human control subjects not having colorectal cancerclassifies the test subject as more likely to have colorectal cancerthan to not have colorectal cancer, and where, for IL2RB, an indicationby the value that the level of RNA encoded by the gene in blood of thetest subject is lower than the level of RNA encoded by the gene in bloodof human control subjects not having colorectal cancer classifies thetest subject as more likely to have colorectal cancer than to not havecolorectal cancer.

Application of computers for provide a classification of a test subjectas more likely to have a disease than to not have the disease, so as toenable the method of the invention, is routinely practiced in the artusing computer systems, and optionally computer-readable media,routinely used in the art.

Thus, according to a further aspect of the invention there is provided acomputer system for providing a classification that a test subject ismore likely to have colorectal cancer than to not have colorectalcancer. The computer system comprises a processor; and a memoryconfigured with instructions that cause the processor to provide a userwith the classification, where the instructions comprise causing theprocessor to apply to test data, and to negative control datarepresenting a level of RNA encoded by the gene in blood of humancontrol subjects not having colorectal cancer, a mathematical formulafor generating a value representing a fold-change between the level ofRNA encoded by the gene in blood of the test subject and the level ofRNA encoded by the gene in blood of human control subjects not havingcolorectal cancer where, for ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 andVNN1, a value indicating that the level of RNA encoded by the gene inblood of the test subject is higher, for example within a range ofsuitable fold-changes taught herein, than the level of RNA encoded bythe gene in blood of human control subjects not having colorectal cancerclassifies the test subject as more likely to have colorectal cancerthan to not have colorectal cancer, and where, for IL2RB, a valueindicating that the level of RNA encoded by the gene in blood of thetest subject is lower, for example within a range of suitablefold-changes disclosed herein, than the level of RNA encoded by the genein blood of human control subjects not having colorectal cancerclassifies the test subject as more likely to have colorectal cancerthan to not have colorectal cancer.

The instructions may be provided to the computer in any one of variousways routinely employed in the art. In one aspect, the instructions areprovided to the computer using a computer-readable medium.

Thus, according to yet another aspect of the invention there is provideda computer-readable medium having instructions stored thereon that areoperable when executed by a computer for applying to test data and tonegative control data representing a level of RNA encoded by a markergene of the invention in blood of human control subjects not havingcolorectal cancer, a mathematical formula for generating a valuerepresenting the fold-change between the level of RNA encoded by thegene in blood of the test subject and the level of RNA encoded by thegene in blood of human control subjects not having colorectal cancer,where, for ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1, a valueindicating that the level of RNA encoded by the gene in blood of thetest subject is higher, for example, within a suitable range offold-changes disclosed herein, than the level of RNA encoded by the genein blood of human control subjects not having colorectal cancerclassifies the test subject as more likely to have colorectal cancerthan to not have colorectal cancer, and where, for IL2RB, a valueindicating that the level of RNA encoded by the gene in blood of thetest subject is lower, for example, within a suitable range offold-changes disclosed herein, than the level of RNA encoded by the genein blood of human control subjects not having colorectal cancerclassifies the test subject as more likely to have colorectal cancerthan to not have colorectal cancer.

Thus, according to still yet another aspect of the invention there isprovided a computer-readable medium having instructions stored thereonthat are operable when executed by a computer for applying, to test datarepresenting a level of RNA encoded by the gene in blood of a human testsubject, and to negative control data representing a level of RNAencoded by the gene in blood of human control subjects not havingcolorectal cancer, a mathematical formula for generating a valueindicating, for ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1, whetherthe level of RNA encoded by the gene in blood of the test subject ishigher than the level of RNA encoded by the gene in blood of humancontrol subjects not having colorectal cancer, and, for IL2RB, whetherthe level of RNA encoded by the gene in blood of the test subject islower than the level of RNA encoded by the gene in blood of humancontrol subjects not having colorectal cancer, where, for ANXA3, CLEC4D,LMNB1, PRRG4, TNFAIP6 and VNN1, an indication by the value that thelevel of RNA encoded by the gene in blood of the test subject is higherthan the level of RNA encoded by the gene in blood of human controlsubjects not having colorectal cancer classifies the test subject asmore likely to have colorectal cancer than to not have colorectalcancer, and where, for IL2RB, an indication by the value that the levelof RNA encoded by the gene in blood of the test subject is lower thanthe level of RNA encoded by the gene in blood of human control subjectsnot having colorectal cancer classifies the test subject as more likelyto have colorectal cancer than to not have colorectal cancer.

An exemplary computer system for practicing certain of the methodsdescribed herein is described in FIG. 1.

FIG. 1 shows a schematic of a general-purpose computer system 100suitable for practicing the methods described herein. The computersystem 100, shown as a self-contained unit but not necessarily solimited, comprises at least one data processing unit (CPU) 102, a memory104, which will typically include both high speed random access memoryas well as non-volatile memory (such as one or more magnetic diskdrives) but may be simply flash memory, a user interface 108, optionallya disk 110 controlled by a disk controller 112, and at least oneoptional network or other communication interface card 114 forcommunicating with other computers as well as other devices. At leastthe CPU 102, memory 104, user interface 108, disk controller wherepresent, and network interface card, communicate with one another via atleast one communication bus 106.

Memory 104 stores procedures and data, typically including: an operatingsystem 140 for providing basic system services; application programs 152such as user level programs for viewing and manipulating data,evaluating formulae for the purpose of diagnosing a test subject;authoring tools for assisting with the writing of computer programs; afile system 142, a user interface controller 144 for handlingcommunications with a user via user interface 108, and optionally one ormore databases 146 for storing data of the invention and otherinformation, optionally a graphics controller 148 for controllingdisplay of data, and optionally a floating point coprocessor 150dedicated to carrying out mathematical operations. The methods of theinvention may also draw upon functions contained in one or moredynamically linked libraries, not shown in FIG. 1, but stored either inMemory 104, or on disk 110, or accessible via network interfaceconnection 114.

User interface 108 may comprise a display 128, a mouse 126, and akeyboard 130. Although shown as separate components in FIG. 1, one ormore of these user interface components can be integrated with oneanother in embodiments such as handheld computers. Display 128 may be acathode ray tube (CRT), or flat-screen display such as an LCD based onactive matrix or TFT embodiments, or may be an electroluminescentdisplay, based on light emitting organic molecules such as conjugatedsmall molecules or polymers. Other embodiments of a user interface notshown in FIG. 1 include, e.g., several buttons on a keypad, acard-reader, a touch-screen with or without a dedicated touching device,a trackpad, a trackball, or a microphone used in conjunction withvoice-recognition software, or any combination thereof, or asecurity-device such as a fingerprint sensor or a retinal scanner thatprohibits an unauthorized user from accessing data and programs storedin system 100.

System 100 may also be connected to an output device such as a printer(not shown), either directly through a dedicated printer cable connectedto a serial or USB port, or wirelessly, or via a network connection.

The database 146 may instead, optionally, be stored on disk 110 incircumstances where the amount of data in the database is too great tobe efficiently stored in memory 104. The database may also instead, orin part, be stored on one or more remote computers that communicate withcomputer system 100 through network interface connection 114.

The network interface 134 may be a connection to the internet or to alocal area network via a cable and modem, or ethernet, firewire, or USBconnectivity, or a digital subscriber line. Preferably the computernetwork connection is wireless, e.g., utilizing CDMA, GSM, or GPRS, orbluetooth, or standards such as 802.11a, 802.11b, or 802.11g.

It would be understood that various embodiments and configurations anddistributions of the components of system 10 across different devicesand locations are consistent with practice of the methods describedherein. For example, a user may use a handheld embodiment that acceptsdata from a test subject, and transmits that data across a networkconnection to another device or location where the data is analyzedaccording to a formulae described herein. A result of such an analysiscan be stored at the other location and/or additionally transmitted backto the handheld embodiment. In such a configuration, the act ofaccepting data from a test subject can include the act of a userinputting the information. The network connection can include aweb-based interface to a remote site at, for example, a healthcareprovider. Alternatively, system 10 can be a device such as a handhelddevice that accepts data from the test subject, analyzes the data, suchas by inputting the data into a formula as further described herein, andgenerating a result that is displayed to the user. The result can thenbe, optionally, transmitted back to a remote location via a networkinterface such as a wireless interface. System 100 may further beconfigured to permit a user to transmit by e-mail results of an analysisdirectly to some other party, such as a healthcare provider, or adiagnostic facility, or a patient.

In one aspect of the invention there is provided a method of determiningwhether a subject is at an increased risk of having colorectal cancerrelative to the general population. The method comprises obtaining atest biological sample of blood from the subject; for each of a set ofgenes selected from the group consisting of ANXA3, CLEC4D, LMNB1, PRRG4,TNFAIP6 and VNN1, determining the amount of RNA encoded by the gene inthe test biological sample; comparing the determined amount of RNA foreach these genes with the amount in one or more control biologicalsamples of blood; and concluding or determining that the subject is atincreased risk, average risk or decreased risk of having colorectalcancer relative to the general population if the amount of RNA encodedby each gene in the test biological sample is higher than in the controlbiological samples for genes ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 andVNN1, and lower for IL2RB.

A test subject would be considered as being at “increased risk” ofhaving or developing colorectal cancer if the amount of RNA encoded byANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and/or VNN1 present in the testbiological sample is higher than that seen in the control samples to anapproximate extent (plus or minus 10%) seen in the working examplesherein. A test subject would be considered as being at “increased risk”of having or developing colorectal cancer if the amount of RNA encodedby IL2RB present in the test biological sample is lower than that seenin the control samples to an approximate extent (plus or minus 10%) seenin the working examples herein.

A combination of marker genes of the invention, such as ANXA3, CLEC4D,LMNB1, PRRG4, VNN1, and IL2RB, can be used together with the known CRCprevalence rate to determine useful thresholds for stratifying theprobability of having colorectal cancer in an average risk population.Using the combined training/blind set (IL2RB duplex) described in theExamples, an increased probability threshold can be selected to identifya sub-population with a colorectal cancer occurrence rate of 1.5%, a3-fold increase over the base disease prevalence rate; this thresholdreflects the same relative risk associated with having a first degreerelative with colorectal cancer. A decreased probability thresholdreflecting a sensitivity for colorectal cancer detection of, forexample, 80%, 75%, 70%, 65%, can be selected to identify alower-than-average probability sub-population. This approach can be usedto stratify patients into an increased probability group, a decreasedprobability, and an average probability group.

One of ordinary skill in the art will be able to determine directly fromthe literature, or will be able to calculate from available statisticaldata, a suitable prevalence rate of colorectal cancer for practicingembodiments of the invention. For example, the prevalence rate forcolorectal cancer in the average risk population over 50 years of agehas been determined to be 0.7% (see for example Imperiale T F. et al.,2004. Colorectal Cancer Study Group. Fecal DNA versus fecal occult bloodfor colorectal-cancer screening in an average-risk population. New EnglJ Med 351:2704-14).

It will be appreciated that components for practicing quantitative PCRaccording to the method of the invention may be assembled in a kit.

Thus, according to still another aspect of the invention there isprovided a kit. The kit comprises packaging and contains, for each geneof a set of two or more of the following target genes of the invention:ACTB, ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1; a primer setcapable of generating an amplification product of DNA complementary toRNA encoded, in a human subject, only by the gene.

In various aspects of the kit of the invention, the set of genes may beany combination of two or more of the target genes of the invention, asdescribed hereinabove and in the Examples section, below.

In one aspect of the invention, the kit further contains two or more ofthe following components: a thermostable polymerase, a reversetranscriptase, deoxynucleotide triphosphates, nucleotide triphosphatesand enzyme buffer.

In another aspect of the invention, the kit further contains at leastone labeled probe capable of selectively hybridizing to either a senseor an antisense strand of the amplification product.

In yet another aspect of the invention, the kit further contains acomputer-readable medium of the invention.

In one aspect, the kit is identified in print in or on the packaging asbeing for determining a probability that a test subject has colorectalcancer, for example, a probability that a test subject has colorectalcancer as opposed to not having colorectal cancer.

In another aspect, the kit is identified in print in or on the packagingas being for classifying a test subject as being more likely to havecolorectal cancer than to not have colorectal cancer, and/or as beingmore likely to not have colorectal cancer than to have colorectalcancer.

In a further aspect, the kit is identified in print in or on thepackaging as being for determining whether a test subject is at anincreased risk of having colorectal cancer relative to the generalpopulation

In various aspects of the kit of the invention, the set of genes may beany combination of two or more of the target genes of the invention.

Sets of genes of the invention which consist of two or more of ACTB,ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1 include: ACTB,ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6, VNN1; ACTB, ANXA3, CLEC4D,IL2RB, LMNB1, PRRG4, VNN1; ACTB, ANXA3, CLEC4D, IL2RB, PRRG4; ACTB,ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4; ACTB, ANXA3, CLEC4D, IL2RB, PRRG4,VNN1; ACTB, ANXA3, IL2RB, LMNB1, PRRG4, VNN1; ACTB, ANXA3, CLEC4D,IL2RB, PRRG4, TNFAIP6; ACTB, ANXA3, IL2RB, LMNB1, PRRG4, TNFAIP6; ACTB,ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6; ACTB, ANXA3, CLEC4D, IL2RB,PRRG4, TNFAIP6, VNN1; ACTB, ANXA3, IL2RB, LMNB1, PRRG4, TNFAIP6, VNN1;ACTB, ANXA3, IL2RB, LMNB1, PRRG4; ACTB, IL2RB, PRRG4, VNN1; ACTB, ANXA3,IL2RB, PRRG4, VNN1; ACTB, CLEC4D, IL2RB, PRRG4, VNN1; ACTB, IL2RB,LMNB1, PRRG4, VNN1; ACTB, CLEC4D, IL2RB, LMNB1, PRRG4, VNN1; ACTB,ANXA3, IL2RB, PRRG4, TNFAIP6; ACTB, IL2RB, PRRG4, TNFAIP6, VNN1; ACTB,ANXA3, IL2RB, PRRG4, TNFAIP6, VNN1; ACTB, CLEC4D, IL2RB, PRRG4, TNFAIP6,VNN1; ACTB, IL2RB, LMNB1, PRRG4, TNFAIP6, VNN1; ACTB, CLEC4D, IL2RB,LMNB1, PRRG4, TNFAIP6, VNN1; ACTB, IL2RB, PRRG4; ACTB, ANXA3, IL2RB,PRRG4; ACTB, CLEC4D, IL2RB, PRRG4; ACTB, IL2RB, LMNB1, PRRG4; ACTB,CLEC4D, IL2RB, LMNB1, PRRG4; ACTB, IL2RB, PRRG4, TNFAIP6; ACTB, CLEC4D,IL2RB, PRRG4, TNFAIP6; ACTB, IL2RB, LMNB1, PRRG4, TNFAIP6; ACTB, CLEC4D,IL2RB, LMNB1, PRRG4, TNFAIP6; ACTB, ANXA3, IL2RB, VNN1; ACTB, ANXA3,CLEC4D, IL2RB, VNN1; ACTB, ANXA3, IL2RB, LMNB1, VNN1; ACTB, ANXA3,CLEC4D, IL2RB, LMNB1, VNN1; ACTB, ANXA3, CLEC4D, LMNB1, PRRG4, VNN1;ACTB, ANXA3, IL2RB, TNFAIP6, VNN1; ACTB, ANXA3, CLEC4D, IL2RB, TNFAIP6,VNN1; ACTB, ANXA3, IL2RB, LMNB1, TNFAIP6, VNN1; ACTB, ANXA3, CLEC4D,IL2RB, LMNB1, TNFAIP6, VNN1; ACTB, ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6,VNN1; ACTB, ANXA3, IL2RB; ACTB, ANXA3, CLEC4D, IL2RB; ACTB, ANXA3,IL2RB, LMNB1; ACTB, ANXA3, CLEC4D, IL2RB, LMNB1; ACTB, ANXA3, CLEC4D,LMNB1, PRRG4; ACTB, CLEC4D, IL2RB, LMNB1, VNN1; ACTB, ANXA3, IL2RB,TNFAIP6; ACTB, ANXA3, CLEC4D, IL2RB, TNFAIP6; ACTB, ANXA3, IL2RB, LMNB1,TNFAIP6; ACTB, ANXA3, CLEC4D, IL2RB, LMNB1, TNFAIP6; ACTB, ANXA3,CLEC4D, LMNB1, PRRG4, TNFAIP6; ACTB, IL2RB, LMNB1, TNFAIP6, VNN1; ACTB,CLEC4D, IL2RB, LMNB1, TNFAIP6, VNN1; ACTB, IL2RB, LMNB1, VNN1; ACTB,ANXA3, LMNB1, PRRG4, VNN1; ACTB, ANXA3, LMNB1, PRRG4, TNFAIP6, VNN1;ACTB, ANXA3, CLEC4D, PRRG4; ACTB, ANXA3, LMNB1, PRRG4; ACTB, CLEC4D,IL2RB, VNN1; ACTB, ANXA3, CLEC4D, PRRG4, VNN1; ACTB, IL2RB, LMNB1,TNFAIP6; ACTB, CLEC4D, IL2RB, LMNB1, TNFAIP6; ACTB, ANXA3, CLEC4D,PRRG4, TNFAIP6; ACTB, ANXA3, LMNB1, PRRG4, TNFAIP6; ACTB, IL2RB,TNFAIP6, VNN1; ACTB, CLEC4D, IL2RB, TNFAIP6, VNN1; ACTB, ANXA3, CLEC4D,PRRG4, TNFAIP6, VNN1; ACTB, IL2RB, LMNB1; ACTB, CLEC4D, IL2RB, LMNB1;ACTB, IL2RB, VNN1; ACTB, ANXA3, CLEC4D, LMNB1, VNN1; ACTB, ANXA3,CLEC4D, LMNB1, TNFAIP6, VNN1; ACTB, ANXA3, CLEC4D, LMNB1; ACTB, ANXA3,PRRG4; ACTB, ANXA3, CLEC4D, VNN1; ACTB, ANXA3, LMNB1, VNN1; ACTB, ANXA3,PRRG4, VNN1; ACTB, ANXA3, CLEC4D, LMNB1, TNFAIP6; ACTB, ANXA3, PRRG4,TNFAIP6; ACTB, ANXA3, CLEC4D, TNFAIP6, VNN1; ACTB, ANXA3, LMNB1,TNFAIP6, VNN1; ACTB, ANXA3, PRRG4, TNFAIP6, VNN1; ACTB, ANXA3; ACTB,ANXA3, CLEC4D; ACTB, ANXA3, LMNB1; ACTB, ANXA3, VNN1; ACTB, ANXA3,TNFAIP6; ACTB, ANXA3, CLEC4D, TNFAIP6; ACTB, IL2RB, TNFAIP6; ACTB,CLEC4D, IL2RB, TNFAIP6; ACTB, ANXA3, LMNB1, TNFAIP6; ACTB, ANXA3,TNFAIP6, VNN1; ACTB, CLEC4D, IL2RB; ACTB, PRRG4, VNN1; ACTB, CLEC4D,PRRG4, VNN1; ACTB, LMNB1, PRRG4, VNN1; ACTB, CLEC4D, LMNB1, PRRG4, VNN1;ACTB, PRRG4, TNFAIP6, VNN1; ACTB, CLEC4D, PRRG4, TNFAIP6, VNN1; ACTB,LMNB1, PRRG4, TNFAIP6, VNN1; ACTB, CLEC4D, LMNB1, PRRG4, TNFAIP6, VNN1;ACTB, PRRG4; ACTB, CLEC4D, PRRG4; ACTB, LMNB1, PRRG4; ACTB, CLEC4D,LMNB1, PRRG4; ACTB, PRRG4, TNFAIP6; ACTB, CLEC4D, PRRG4, TNFAIP6; ACTB,LMNB1, PRRG4, TNFAIP6; ACTB, CLEC4D, LMNB1, PRRG4, TNFAIP6; ACTB, LMNB1,TNFAIP6, VNN1; ACTB, CLEC4D, VNN1; ACTB, LMNB1, VNN1; ACTB, CLEC4D,LMNB1, VNN1; ACTB, LMNB1, TNFAIP6; ACTB, LMNB1, TNFAIP6; ACTB, TNFAIP6,VNN1; ACTB, CLEC4D, TNFAIP6, VNN1; ACTB, CLEC4D, LMNB1, TNFAIP6, VNN1;ACTB, LMNB1; ACTB, CLEC4D, LMNB1; ACTB, VNN1; ACTB, CLEC4D, TNFAIP6;ACTB, TNFAIP6; ACTB, CLEC4D; and ACTB, IL2RB.

In one aspect of the kit of the invention, the set of one or more genesconsists of a housekeeping gene such as ACTB, and one or more of thecolorectal cancer marker genes: ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4,TNFAIP6 and VNN1.

In one aspect of the kit of the invention, the set of one or more genesconsists of ACTB and ANXA3.

In one aspect of the kit of the invention, the set of one or more genesconsists of ACTB and CLEC4D.

In one aspect of the kit of the invention, the set of one or more genesconsists of ACTB and IL2RB.

In one aspect of the kit of the invention, the set of one or more genesconsists of ACTB and LMNB1.

In one aspect of the kit of the invention, the set of one or more genesconsists of ACTB and PRRG4.

In one aspect of the kit of the invention, the set of one or more genesconsists of ACTB and TNFAIP6.

In one aspect of the kit of the invention, the set of one or more genesconsists of ACTB and VNN1.

In another aspect of the kit of the invention, the set of one or moregenes consists of IL2RB, and one or more of the colorectal cancer markergenes: ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1.

In one aspect of the kit of the invention, the set of one or more genesconsists of IL2RB and ANXA3.

In one aspect of the kit of the invention, the set of one or more genesconsists of IL2RB and CLEC4D.

In one aspect of the kit of the invention, the set of one or more genesconsists of IL2RB and LMNB1.

In one aspect of the kit of the invention, the set of one or more genesconsists of IL2RB and PRRG4.

In one aspect of the kit of the invention, the set of one or more genesconsists of IL2RB and TNFAIP6.

In one aspect of the kit of the invention, the set of one or more genesconsists of IL2RB and VNN1.

In one aspect of the invention, the kit contains a primer having anucleotide sequence identified as SEQ ID NO: 1, and a primer having anucleotide sequence identified as SEQ ID NO: 2.

In one aspect of the invention, the kit contains a primer having anucleotide sequence identified as SEQ ID NO: 1, and a primer having anucleotide sequence identified as SEQ ID NO: 2 and the kit furthercontains a labeled probe which comprises a polynucleotide having anucleic acid sequence identified as SEQ ID NO: 3.

In one aspect of the invention, the kit contains a primer having anucleotide sequence identified as SEQ ID NO: 10, and a primer having anucleotide sequence identified as SEQ ID NO: 11.

In one aspect of the invention, the kit contains a primer having anucleotide sequence identified as SEQ ID NO: 10, and a primer having anucleotide sequence identified as SEQ ID NO: 11, and the kit furthercontains a labeled probe which comprises a polynucleotide having anucleic acid sequence identified as SEQ ID NO: 12.

In one aspect of the invention, the kit contains a primer having anucleotide sequence identified as SEQ ID NO: 19, and a primer having anucleotide sequence identified as SEQ ID NO: 20.

In one aspect of the invention, the kit contains a primer having anucleotide sequence identified as SEQ ID NO: 19, and a primer having anucleotide sequence identified as SEQ ID NO: 20 and the kit furthercontains a labeled probe which comprises a polynucleotide having anucleic acid sequence identified as SEQ ID NO: 21.

In one aspect of the invention, for example, the kit contains a primerhaving a nucleotide sequence identified as SEQ ID NO: 28, and a primerhaving a nucleotide sequence identified as SEQ ID NO: 29.

In one aspect of the invention, for example, the kit contains a primerhaving a nucleotide sequence identified as SEQ ID NO: 28, and a primerhaving a nucleotide sequence identified as SEQ ID NO: 29 and the kitfurther contains a labeled probe which comprises a polynucleotide havinga nucleic acid sequence identified as SEQ ID NO: 30.

In one aspect of the invention, the kit contains a primer having anucleotide sequence identified as SEQ ID NO: 37, and a primer having anucleotide sequence identified as SEQ ID NO: 38.

In one aspect of the invention, the kit contains a primer having anucleotide sequence identified as SEQ ID NO: 37, and a primer having anucleotide sequence identified as SEQ ID NO: 38 and the kit furthercontains a labeled probe which comprises a polynucleotide having anucleic acid sequence identified as SEQ ID NO: 39.

In one aspect of the invention, the kit contains a primer having anucleotide sequence identified as SEQ ID NO: 46, and a primer having anucleotide sequence identified as SEQ ID NO: 47.

In one aspect of the invention, the kit contains a primer having anucleotide sequence identified as SEQ ID NO: 46, and a primer having anucleotide sequence identified as SEQ ID NO: 47, and the kit furthercontains a labeled probe which comprises a polynucleotide having anucleic acid sequence identified as SEQ ID NO: 48.

In one aspect of the invention, the kit contains a primer having anucleotide sequence identified as SEQ ID NO: 55, and a primer having anucleotide sequence identified as SEQ ID NO: 56.

In one aspect of the invention, the kit contains a primer having anucleotide sequence identified as SEQ ID NO: 55, and a primer having anucleotide sequence identified as SEQ ID NO: 56 and the kit furthercontains a labeled probe which comprises a polynucleotide having anucleic acid sequence identified as SEQ ID NO: 57.

Further, non-limiting, specific aspects of the invention include thefollowing:

One aspect of the invention disclosed herein is a method of determininga probability that a human test subject has colorectal cancer as opposedto not having colorectal cancer, the method comprising: the steps of (a)determining a level of RNA encoded by a ANXA3 gene in blood of the testsubject, thereby generating test data; (b) providing positive controldata representing levels of RNA encoded by the gene in blood of humancontrol subjects having colorectal cancer, and providing negativecontrol data representing levels of RNA encoded by the gene in blood ofhuman control subjects not having colorectal cancer; and (c) determininga probability that the test data corresponds to the positive controldata and not to the negative control data, where the probability thatthe test data corresponds to the positive control data and not to thenegative control data represents the probability that the test subjecthas colorectal cancer as opposed to not having colorectal cancer.Another aspect of the invention disclosed herein is a method ofdetermining a probability that a human test subject has colorectalcancer as opposed to not having colorectal cancer, the method comprisingthe steps of (a) determining a level of RNA encoded by a CLEC4D gene inblood of the test subject, thereby generating test data; (b) providingpositive control data representing levels of RNA encoded by the gene inblood of human control subjects having colorectal cancer, and providingnegative control data representing levels of RNA encoded by the gene inblood of human control subjects not having colorectal cancer; and (c)determining a probability that the test data corresponds to the positivecontrol data and not to the negative control data, where the probabilitythat the test data corresponds to the positive control data and not tothe negative control data represents the probability that the testsubject has colorectal cancer as opposed to not having colorectalcancer. Another aspect of the invention disclosed herein is a method ofdetermining a probability that a human test subject has colorectalcancer as opposed to not having colorectal cancer, the method comprisingthe steps of (a) determining a level of RNA encoded by a IL2RB gene inblood of the test subject, thereby generating test data; (b) providingpositive control data representing levels of RNA encoded by the gene inblood of human control subjects having colorectal cancer, and providingnegative control data representing levels of RNA encoded by the gene inblood of human control subjects not having colorectal cancer; and (c)determining a probability that the test data corresponds to the positivecontrol data and not to the negative control data, where the probabilitythat the test data corresponds to the positive control data and not tothe negative control data represents the probability that the testsubject has colorectal cancer as opposed to not having colorectalcancer. Another aspect of the invention disclosed herein is a method ofdetermining a probability that a human test subject has colorectalcancer as opposed to not having colorectal cancer, the method comprisingthe steps of: (a) determining a level of RNA encoded by a LMNB1 gene inblood of the test subject, thereby generating test data; (b) providingpositive control data representing levels of RNA encoded by the gene inblood of human control subjects having colorectal cancer, and providingnegative control data representing levels of RNA encoded by the gene inblood of human control subjects not having colorectal cancer; and (c)determining a probability that the test data corresponds to the positivecontrol data and not to the negative control data, where the probabilitythat the test data corresponds to the positive control data and not tothe negative control data represents the probability that the testsubject has colorectal cancer as opposed to not having colorectalcancer. Another aspect of the invention disclosed herein is a method ofdetermining a probability that a human test subject has colorectalcancer as opposed to not having colorectal cancer, the method comprisingthe steps of: (a) determining a level of RNA encoded by a PRRG4 gene inblood of the test subject, thereby generating test data; (b) providingpositive control data representing levels of RNA encoded by the gene inblood of human control subjects having colorectal cancer, and providingnegative control data representing levels of RNA encoded by the gene inblood of human control subjects not having colorectal cancer; and (c)determining a probability that the test data corresponds to the positivecontrol data and not to the negative control data, where the probabilitythat the test data corresponds to the positive control data and not tothe negative control data represents the probability that the testsubject has colorectal cancer as opposed to not having colorectalcancer. Another aspect of the invention disclosed herein is a method ofdetermining a probability that a human test subject has colorectalcancer as opposed to not having colorectal cancer, the method comprisingthe steps of: (a) determining a level of RNA encoded by a TNFAIP6 genein blood of the test subject, thereby generating test data; (b)providing positive control data representing levels of RNA encoded bythe gene in blood of human control subjects having colorectal cancer,and providing negative control data representing levels of RNA encodedby the gene in blood of human control subjects not having colorectalcancer; and (c) determining a probability that the test data correspondsto the positive control data and not to the negative control data, wherethe probability that the test data corresponds to the positive controldata and not to the negative control data represents the probabilitythat the test subject has colorectal cancer as opposed to not havingcolorectal cancer. Another aspect of the invention disclosed herein is amethod of determining a probability that a human test subject hascolorectal cancer as opposed to not having colorectal cancer, the methodcomprising the steps of: (a) determining a level of RNA encoded by aVNN1 gene in blood of the test subject, thereby generating test data;(b) providing positive control data representing levels of RNA encodedby the gene in blood of human control subjects having colorectal cancer,and providing negative control data representing levels of RNA encodedby the gene in blood of human control subjects not having colorectalcancer; and (c) determining a probability that the test data correspondsto the positive control data and not to the negative control data, wherethe probability that the test data corresponds to the positive controldata and not to the negative control data represents the probabilitythat the test subject has colorectal cancer as opposed to not havingcolorectal cancer.

An embodiment of aspects of the invention disclosed herein includes thatthe determining of the level of RNA encoded by the gene in blood of thetest subject be effected by determining the level of RNA encoded by thegene in a blood sample isolated from the test subject. An embodiment ofaspects of the invention disclosed herein includes the further step ofdetermining the levels of RNA encoded by the gene in blood of apopulation of human subjects having colorectal cancer, thereby providingthe positive control data representing the levels of RNA encoded by thegene in blood of human control subjects having colorectal cancer, anddetermining levels of RNA encoded by the gene in blood of a populationof human subjects not having colorectal cancer, thereby providing thenegative control data representing the levels of RNA encoded by the genein blood of human control subjects not having colorectal cancer. Anembodiment of aspects of the invention disclosed herein includes thatthe level of RNA encoded by the gene in blood of the test subject isdetermined via quantitative reverse transcriptase-polymerase chainreaction analysis. An embodiment of aspects of the invention disclosedherein includes that the level of RNA encoded by the gene in blood ofthe test subject and the levels of RNA encoded by the gene in blood ofthe control subjects are determined via the same method. An embodimentof aspects of the invention disclosed herein includes that thedetermining of the probability that the test data corresponds to thepositive control data and not to the negative control data is effectedby applying to the test data a mathematical model derived from thepositive control data and from the negative control data, and where themathematical model is for determining the probability that datarepresenting a level of RNA encoded by the gene corresponds to thepositive control data and not to the negative control data. Anembodiment of aspects of the invention disclosed herein includes thatthe level of RNA encoded by the gene in blood of the test subject isdetermined as a ratio to a level of RNA encoded by ACTB in blood of thetest subject. An aspect of this latter embodiment includes that thelevel of RNA encoded by the gene in blood of the test subject and thelevel of RNA encoded by ACTB in blood of the test subject are determinedvia duplex quantitative reverse transcriptase-polymerase chain reactionanalysis of RNA encoded by the gene and of RNA encoded by ACTB. Anembodiment of aspects of the invention disclosed herein includes thatthe level of RNA encoded by the gene in blood of the test subject isdetermined as a ratio to a level of RNA encoded by IL2RB in blood of thetest subject. An aspect of this latter embodiment includes that thelevel of RNA encoded by the gene in blood of the test subject and thelevel of RNA encoded by IL2RB in blood of the test subject aredetermined via duplex quantitative reverse transcriptase-polymerasechain reaction analysis of RNA encoded by the gene and of RNA encoded byIL2RB.

An aspect of the invention disclosed herein is a computer-based methodof determining a probability that a human test subject has colorectalcancer as opposed to not having colorectal cancer, from test datarepresenting a level of RNA encoded by a ANXA3 gene in blood of the testsubject, the method comprising computer-implemented steps of: (a)applying to the test data a mathematical model derived from positivecontrol data representing levels of RNA encoded by the gene in blood ofhuman control subjects having colorectal cancer, and from negativecontrol data representing levels of RNA encoded by the gene in blood ofhuman control subjects not having colorectal cancer, where themathematical model is for determining a probability that datarepresenting a level of RNA encoded by the gene corresponds to thepositive control data and not to the negative control data; and (b)outputting the probability that data representing a level of RNA encodedby the gene corresponds to the positive control data and not to thenegative control data, where the probability that the test datacorresponds to the positive control data and not to the negative controldata represents the probability that the test subject has colorectalcancer as opposed to not having colorectal cancer. Another aspect of theinvention disclosed herein is a computer-based method of determining aprobability that a human test subject has colorectal cancer as opposedto not having colorectal cancer, from test data representing a level ofRNA encoded by a ANXA3 gene in blood of the test subject, the methodcomprising computer-implemented steps of: inputting, to a computer, testdata representing a level of RNA encoded by a CLEC4D gene in blood ofthe test subject; and causing the computer to apply to the test data amathematical model derived from positive control data representinglevels of RNA encoded by the gene in blood of human control subjectshaving colorectal cancer, and from negative control data representinglevels of RNA encoded by the gene in blood of human control subjects nothaving colorectal cancer, where the mathematical model is fordetermining a probability that data representing a level of RNA encodedby the gene corresponds to the positive control data and not to thenegative control data; and (b) outputting the probability that datarepresenting a level of RNA encoded by the gene corresponds to thepositive control data and not to the negative control data, where theprobability that the test data corresponds to the positive control dataand not to the negative control data represents the probability that thetest subject has colorectal cancer as opposed to not having colorectalcancer. Another aspect of the invention disclosed herein is acomputer-based method of determining a probability that a human testsubject has colorectal cancer as opposed to not having colorectalcancer, from test data representing a level of RNA encoded by a ANXA3gene in blood of the test subject, the method comprisingcomputer-implemented steps of: inputting, to a computer, test datarepresenting a level of RNA encoded by a IL2RB gene in blood of the testsubject; and causing the computer to apply to the test data amathematical model derived from positive control data representinglevels of RNA encoded by the gene in blood of human control subjectshaving colorectal cancer, and from negative control data representinglevels of RNA encoded by the gene in blood of human control subjects nothaving colorectal cancer, where the mathematical model is fordetermining a probability that data representing a level of RNA encodedby the gene corresponds to the positive control data and not to thenegative control data; and (b) outputting the probability that datarepresenting a level of RNA encoded by the gene corresponds to thepositive control data and not to the negative control data, where theprobability that the test data corresponds to the positive control dataand not to the negative control data represents the probability that thetest subject has colorectal cancer as opposed to not having colorectalcancer. Another aspect of the invention disclosed herein is acomputer-based method of determining a probability that a human testsubject has colorectal cancer as opposed to not having colorectalcancer, from test data representing a level of RNA encoded by a ANXA3gene in blood of the test subject, the method comprisingcomputer-implemented steps of: (a) applying to the test data amathematical model derived from positive control data representinglevels of RNA encoded by the gene in blood of human control subjectshaving colorectal cancer, and from negative control data representinglevels of RNA encoded by the gene in blood of human control subjects nothaving colorectal cancer, where the mathematical model is fordetermining a probability that data representing a level of RNA encodedby the gene corresponds to the positive control data and not to thenegative control data; and (b) outputting the probability that datarepresenting a level of RNA encoded by the gene corresponds to thepositive control data and not to the negative control data, where theprobability that the test data corresponds to the positive control dataand not to the negative control data represents the probability that thetest subject has colorectal cancer as opposed to not having colorectalcancer. Another aspect of the invention disclosed herein is acomputer-based method of determining a probability that a human testsubject has colorectal cancer as opposed to not having colorectalcancer, from test data representing a level of RNA encoded by a ANXA3gene in blood of the test subject, the method comprisingcomputer-implemented steps of: (a) applying to the test data amathematical model derived from positive control data representinglevels of RNA encoded by the gene in blood of human control subjectshaving colorectal cancer, and from negative control data representinglevels of RNA encoded by the gene in blood of human control subjects nothaving colorectal cancer, where the mathematical model is fordetermining a probability that data representing a level of RNA encodedby the gene corresponds to the positive control data and not to thenegative control data; and (b) outputting the probability that datarepresenting a level of RNA encoded by the gene corresponds to thepositive control data and not to the negative control data, where theprobability that the test data corresponds to the positive control dataand not to the negative control data represents the probability that thetest subject has colorectal cancer as opposed to not having colorectalcancer. Another aspect of the invention disclosed herein is acomputer-based method of determining a probability that a human testsubject has colorectal cancer as opposed to not having colorectalcancer, from test data representing a level of RNA encoded by a ANXA3gene in blood of the test subject, the method comprisingcomputer-implemented steps of: (a) applying to the test data amathematical model derived from positive control data representinglevels of RNA encoded by the gene in blood of human control subjectshaving colorectal cancer, and from negative control data representinglevels of RNA encoded by the gene in blood of human control subjects nothaving colorectal cancer, where the mathematical model is fordetermining a probability that data representing a level of RNA encodedby the gene corresponds to the positive control data and not to thenegative control data; and (b) outputting the probability that datarepresenting a level of RNA encoded by the gene corresponds to thepositive control data and not to the negative control data, where theprobability that the test data corresponds to the positive control dataand not to the negative control data represents the probability that thetest subject has colorectal cancer as opposed to not having colorectalcancer. Another aspect of the invention disclosed herein is acomputer-based method of determining a probability that a human testsubject has colorectal cancer as opposed to not having colorectalcancer, from test data representing a level of RNA encoded by a ANXA3gene in blood of the test subject, the method comprisingcomputer-implemented steps of: (a) applying to the test data amathematical model derived from positive control data representinglevels of RNA encoded by the gene in blood of human control subjectshaving colorectal cancer, and from negative control data representinglevels of RNA encoded by the gene in blood of human control subjects nothaving colorectal cancer, where the mathematical model is fordetermining a probability that data representing a level of RNA encodedby the gene corresponds to the positive control data and not to thenegative control data; and (b) outputting the probability that datarepresenting a level of RNA encoded by the gene corresponds to thepositive control data and not to the negative control data, where theprobability that the test data corresponds to the positive control dataand not to the negative control data represents the probability that thetest subject has colorectal cancer as opposed to not having colorectalcancer.

An embodiment of the invention's computer based methods includes wherethe level of RNA encoded by the gene in blood of the test subject isdetermined via quantitative reverse transcriptase-polymerase chainreaction analysis. An embodiment of computer based methods of theinvention includes where the level of RNA encoded by the gene in bloodof the test subject and the levels of RNA encoded by the gene in bloodof the control subjects are determined via the same method. Anembodiment of each of computer based methods of the invention includeswhere the level of RNA encoded by the gene in blood of the test subjectis determined as a ratio to a level of RNA encoded by ACTB in blood ofthe test subject. An embodiment of computer based methods of theinvention includes where the level of RNA encoded by the gene in bloodof the test subject and the level of RNA encoded by ACTB in blood of thetest subject are determined via duplex quantitative reversetranscriptase-polymerase chain reaction analysis of RNA encoded by thegene and of RNA encoded by ACTB. An embodiment of each of the computerbased methods of the invention includes where the level of RNA encodedby the gene in blood of the test subject is determined as a ratio to alevel of RNA encoded by IL2RB in blood of the test subject. In a furtherembodiment the level of RNA encoded by the gene in blood of the testsubject and the level of RNA encoded by IL2RB in blood of the testsubject are determined via duplex quantitative reversetranscriptase-polymerase chain reaction analysis of RNA encoded by thegene and of RNA encoded by IL2RB.

Another aspect of the invention disclosed herein is a method ofdetermining a probability that a human test subject has colorectalcancer as opposed to not having colorectal cancer, the methodcomprising, for each gene of a set of one or more genes selected fromthe group consisting of ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 andVNN1: comprising the steps of: (a) determining a level of RNA encoded bythe gene in blood of the test subject, thereby generating test data; (b)providing positive control data representing levels of RNA encoded bythe gene in blood of human control subjects having colorectal cancer,and providing negative control data representing levels of RNA encodedby the gene in blood of human control subjects not having colorectalcancer; and (c) determining a probability that the test data correspondsto the positive control data and not to the negative control data, wherethe probability that the test data corresponds to the positive controldata and not to the negative control data represents the probabilitythat the test subject has colorectal cancer as opposed to not havingcolorectal cancer. An embodiment of this aspect of the inventiondisclosed herein is where the determining of the level of RNA encoded bythe gene in blood of the test subject is effected by determining thelevel of RNA encoded by the gene in a blood sample isolated from thetest subject. An embodiment of this aspect of the invention disclosedherein further comprises determining levels of RNA encoded by the genein blood of a population of human subjects having colorectal cancer,thereby providing the positive control data representing the levels ofRNA encoded by the gene in blood of human control subjects havingcolorectal cancer, and determining levels of RNA encoded by the gene inblood of a population of human subjects not having colorectal cancer,thereby providing the negative control data representing the levels ofRNA encoded by the gene in blood of human control subjects not havingcolorectal cancer. An embodiment of this aspect of the inventiondisclosed herein is where the level of RNA encoded by the gene in bloodof the test subject is determined via quantitative reversetranscriptase-polymerase chain reaction analysis. An embodiment of thisaspect of the invention disclosed herein is where the level of RNAencoded by the gene in blood of the test subject and the levels of RNAencoded by the gene in blood of the control subjects are determined viathe same method. An embodiment of this aspect of the invention disclosedherein is where the determining of the probability that the test datacorresponds to the positive control data and not to the negative controldata is effected by applying to the test data a mathematical modelderived from the positive control data and from the negative controldata, and where the mathematical model is for determining theprobability that data representing a level of RNA encoded by the genecorresponds to the positive control data and not to the negative controldata. An embodiment of this aspect of the invention disclosed herein iswhere the level of RNA encoded by the gene in blood of the test subjectis determined as a ratio to a level of RNA encoded by ACTB in blood ofthe test subject. In a further embodiment, the level of RNA encoded bythe gene in blood of the test subject and the level of RNA encoded byACTB in blood of the test subject are determined via duplex quantitativereverse transcriptase-polymerase chain reaction analysis of RNA encodedby the gene and of RNA encoded by ACTB. An embodiment of this aspect ofthe invention disclosed herein is where the set of one or more genes isa set of one or more genes selected from the group consisting of ANXA3,CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1, and where the level of RNAencoded by the gene in blood of the test subject is determined as aratio to a level of RNA encoded by IL2RB in blood of the test subject.In a further embodiment, the level of RNA encoded by the gene in bloodof the test subject and the level of RNA encoded by IL2RB in blood ofthe test subject are determined via duplex quantitative reversetranscriptase-polymerase chain reaction analysis of RNA encoded by thegene and of RNA encoded by IL2RB.

Another aspect of the invention disclosed herein is a computer-basedmethod of determining a probability that a human test subject hascolorectal cancer as opposed to not having colorectal cancer, from testdata representing a level of RNA encoded by the gene in blood of thetest subject, the method comprising, for each gene of a set of one ormore genes selected from the group consisting of ANXA3, CLEC4D, IL2RB,LMNB1, PRRG4, TNFAIP6 and VNN1, computer-implemented steps of: (a)applying to the test data a mathematical model derived from positivecontrol data representing levels of RNA encoded by the gene in blood ofhuman control subjects having colorectal cancer, and from negativecontrol data representing levels of RNA encoded by the gene in blood ofhuman control subjects not having colorectal cancer, where themathematical model is for determining a probability that datarepresenting a level of RNA encoded by the gene corresponds to thepositive control data and not to the negative control data; and (b)outputting the probability that data representing a level of RNA encodedby the gene corresponds to the positive control data and not to thenegative control data, where the probability that the test datacorresponds to the positive control data and not to the negative controldata represents the probability that the test subject has colorectalcancer as opposed to not having colorectal cancer. In an embodiment ofthis aspect of the invention disclosed herein is where the level of RNAencoded by the gene in blood of the test subject is determined viaquantitative reverse transcriptase-polymerase chain reaction analysis.In an embodiment of this aspect of the invention disclosed herein iswhere the level of RNA encoded by the gene in blood of the test subjectand the levels of RNA encoded by the gene in blood of the controlsubjects are determined via the same method. In an embodiment of thisaspect of the invention disclosed herein is where the level of RNAencoded by the gene in blood of the test subject is determined as aratio to a level of RNA encoded by ACTB in blood of the test subject. Ina further embodiment of this aspect of the invention disclosed herein iswhere the level of RNA encoded by the gene in blood of the test subjectand the level of RNA encoded by ACTB in blood of the test subject aredetermined via duplex quantitative reverse transcriptase-polymerasechain reaction analysis of RNA encoded by the gene and of RNA encoded byACTB. In an embodiment of this aspect of the invention disclosed hereinis where the set of one or more genes is a set of one or more genesselected from the group consisting of ANXA3, CLEC4D, LMNB1, PRRG4,TNFAIP6 and VNN1, and where the level of RNA encoded by the gene inblood of the test subject is determined as a ratio to a level of RNAencoded by IL2RB in blood of the test subject. In a further embodimentof this aspect of the invention disclosed herein, the level of RNAencoded by the gene in blood of the test subject and the level of RNAencoded by IL2RB in blood of the test subject are determined via duplexquantitative reverse transcriptase-polymerase chain reaction analysis ofRNA encoded by the gene and of RNA encoded by IL2RB. In an embodiment ofthis aspect of the invention disclosed herein is where the set of one ormore genes consists of PRRG4. In an embodiment of this aspect of theinvention disclosed herein is where the set of one or more genesconsists of IL2RB and PRRG4.

Another aspect of the invention disclosed herein is a kit comprisingpackaging and containing, for each gene of a set of two or more genesselected from the group consisting of ACTB, ANXA3, CLEC4D, IL2RB, LMNB1,PRRG4, TNFAIP6 and VNN1, a primer set capable of generating anamplification product of DNA complementary to RNA encoded, in a humansubject, only by the gene. An embodiment of this aspect of the inventiondisclosed herein is where the kit further contains two or morecomponents selected from the group consisting of a thermostablepolymerase, a reverse transcriptase, deoxynucleotide triphosphates,nucleotide triphosphates and enzyme buffer. An embodiment of this aspectof the invention disclosed herein is where the kit further contains atleast one labelled probe capable of selectively hybridizing to either asense or an antisense strand of the amplification product. An embodimentof this aspect of the invention disclosed herein is where the kitfurther contains a computer-readable medium having instructions storedthereon that are operable when executed by a computer for applying amathematical model to test data representing a level of RNA encoded bythe gene in blood of a human test subject, where the mathematical modelis derived from positive control data representing levels of RNA encodedby the gene in blood of human control subjects having colorectal cancer,and from negative control data representing levels of RNA encoded by thegene in blood of human control subjects not having colorectal cancer,where the mathematical model is for determining a probability that datarepresenting a level of RNA encoded by the gene corresponds to thepositive control data and not to the negative control data, and wherethe probability that the test data corresponds to the positive controldata and not to the negative control data represents the probabilitythat the test subject has colorectal cancer as opposed to not havingcolorectal cancer. An embodiment of this aspect of the inventiondisclosed herein is where the set of one or more genes of the kitconsists of ACTB and one or more genes selected from the groupconsisting of ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1. Anembodiment of this aspect of the invention disclosed herein is where theset of one or more genes of one or more genes of the kit consists ofACTB and ANXA3. An embodiment of this aspect of the invention disclosedherein is where the set of one or more genes of one or more genes of thekit consists of ACTB and CLEC4D. An embodiment of this aspect of theinvention disclosed herein is where the set of one or more genes of oneor more genes of the kit consists of ACTB and IL2RB. An embodiment ofthis aspect of the invention disclosed herein is where the set of one ormore genes of one or more genes of the kit consists of ACTB and LMNB1.An embodiment of this aspect of the invention disclosed herein is wherethe set of one or more genes of one or more genes of the kit consists ofACTB and PRRG4. An embodiment of this aspect of the invention disclosedherein is where the set of one or more genes of one or more genes of thekit consists of ACTB and TNFAIP6. An embodiment of this aspect of theinvention disclosed herein is where the set of one or more genes of oneor more genes of the kit consists of ACTB and VNN1. An embodiment ofthis aspect of the invention disclosed herein is where the set of one ormore genes of one or more genes of the kit consists of IL2RB and one ormore genes selected from the group consisting of ANXA3, CLEC4D, LMNB1,PRRG4, TNFAIP6 and VNN1. An embodiment of this aspect of the inventiondisclosed herein is where the set of one or more genes of one or moregenes of the kit consists of IL2RB and ANXA3. An embodiment of thisaspect of the invention disclosed herein is where the set of one or moregenes of one or more genes of the kit consists of \ IL2RB and CLEC4D. Anembodiment of this aspect of the invention disclosed herein is where theset of one or more genes of one or more genes of the kit consists ofIL2RB and LMNB1. An embodiment of this aspect of the invention disclosedherein is where the set of one or more genes of one or more genes of thekit consists of IL2RB and PRRG4. An embodiment of this aspect of theinvention disclosed herein is where the set of one or more genes of oneor more genes of the kit consists of IL2RB and TNFAIP6. An embodiment ofthis aspect of the invention disclosed herein is where the set of one ormore genes of one or more genes of the kit consists of IL2RB and VNN1.

Another aspect of the invention disclosed herein is a method ofclassifying a human test subject as more likely to have colorectalcancer than to not have colorectal cancer, the method comprising: (a)determining a level of RNA encoded by a ANXA3 gene in blood of the testsubject, thereby generating test data; (b) providing negative controldata representing a level of RNA encoded by the gene in blood of humancontrol subjects not having colorectal cancer; and (c) applying to thetest data and to the negative control data a mathematical formula forgenerating a value indicating whether the level of RNA encoded by thegene in blood of the test subject is higher than the level of RNAencoded by the gene in blood of human control subjects not havingcolorectal cancer, where an indication by the value that the level ofRNA encoded by the gene in blood of the test subject is higher than thelevel of RNA encoded by the gene in blood of human control subjects nothaving colorectal cancer classifies the test subject as more likely tohave colorectal cancer than to not have colorectal cancer. Anotheraspect of the invention disclosed herein is a method of classifying ahuman test subject as more likely to have colorectal cancer than to nothave colorectal cancer, the method comprising: (a) determining a levelof RNA encoded by a CLEC4D gene in blood of the test subject, therebygenerating test data; (b) providing negative control data representing alevel of RNA encoded by the gene in blood of human control subjects nothaving colorectal cancer; and (c) applying to the test data and to thenegative control data a mathematical formula for generating a valueindicating whether the level of RNA encoded by the gene in blood of thetest subject is higher than the level of RNA encoded by the gene inblood of human control subjects not having colorectal cancer, where anindication by the value that the level of RNA encoded by the gene inblood of the test subject is higher than the level of RNA encoded by thegene in blood of human control subjects not having colorectal cancerclassifies the test subject as more likely to have colorectal cancerthan to not have colorectal cancer. Another aspect of the inventiondisclosed herein is a method of classifying a human test subject as morelikely to have colorectal cancer than to not have colorectal cancer, themethod comprising: (a) determining a level of RNA encoded by a IL2RBgene in blood of the test subject, thereby generating test data; (b)providing negative control data representing a level of RNA encoded bythe gene in blood of human control subjects not having colorectalcancer; and (c) applying to the test data and to the negative controldata a mathematical formula for generating a value indicating whetherthe level of RNA encoded by the gene in blood of the test subject ishigher than the level of RNA encoded by the gene in blood of humancontrol subjects not having colorectal cancer, where an indication bythe value that the level of RNA encoded by the gene in blood of the testsubject is lower than the level of RNA encoded by the gene in blood ofhuman control subjects not having colorectal cancer classifies the testsubject as more likely to have colorectal cancer than to not havecolorectal cancer. Another aspect of the invention disclosed herein is amethod of classifying a human test subject as more likely to havecolorectal cancer than to not have colorectal cancer, the methodcomprising: (a) determining a level of RNA encoded by a LMNB1 gene inblood of the test subject, thereby generating test data; (b) providingnegative control data representing a level of RNA encoded by the gene inblood of human control subjects not having colorectal cancer; and (c)applying to the test data and to the negative control data amathematical formula for generating a value indicating whether the levelof RNA encoded by the gene in blood of the test subject is higher thanthe level of RNA encoded by the gene in blood of human control subjectsnot having colorectal cancer, where an indication by the value that thelevel of RNA encoded by the gene in blood of the test subject is higherthan the level of RNA encoded by the gene in blood of human controlsubjects not having colorectal cancer classifies the test subject asmore likely to have colorectal cancer than to not have colorectalcancer. Another aspect of the invention disclosed herein is a method ofclassifying a human test subject as more likely to have colorectalcancer than to not have colorectal cancer, the method comprising: (a)determining a level of RNA encoded by a PRRG4 gene in blood of the testsubject, thereby generating test data; (b) providing negative controldata representing a level of RNA encoded by the gene in blood of humancontrol subjects not having colorectal cancer; and (c) applying to thetest data and to the negative control data a mathematical formula forgenerating a value indicating whether the level of RNA encoded by thegene in blood of the test subject is higher than the level of RNAencoded by the gene in blood of human control subjects not havingcolorectal cancer, where an indication by the value that the level ofRNA encoded by the gene in blood of the test subject is higher than thelevel of RNA encoded by the gene in blood of human control subjects nothaving colorectal cancer classifies the test subject as more likely tohave colorectal cancer than to not have colorectal cancer. Anotheraspect of the invention disclosed herein is a method of classifying ahuman test subject as more likely to have colorectal cancer than to nothave colorectal cancer, the method comprising: (a) determining a levelof RNA encoded by a TNFAIP6 gene in blood of the test subject, therebygenerating test data; (b) providing negative control data representing alevel of RNA encoded by the gene in blood of human control subjects nothaving colorectal cancer; and (c) applying to the test data and to thenegative control data a mathematical formula for generating a valueindicating whether the level of RNA encoded by the gene in blood of thetest subject is higher than the level of RNA encoded by the gene inblood of human control subjects not having colorectal cancer, where anindication by the value that the level of RNA encoded by the gene inblood of the test subject is higher than the level of RNA encoded by thegene in blood of human control subjects not having colorectal cancerclassifies the test subject as more likely to have colorectal cancerthan to not have colorectal cancer. Another aspect of the inventiondisclosed herein is a method of classifying a human test subject as morelikely to have colorectal cancer than to not have colorectal cancer, themethod comprising: (a) determining a level of RNA encoded by a VNN1 genein blood of the test subject, thereby generating test data; (b)providing negative control data representing a level of RNA encoded bythe gene in blood of human control subjects not having colorectalcancer; and (c) applying to the test data and to the negative controldata a mathematical formula for generating a value indicating whetherthe level of RNA encoded by the gene in blood of the test subject ishigher than the level of RNA encoded by the gene in blood of humancontrol subjects not having colorectal cancer, where an indication bythe value that the level of RNA encoded by the gene in blood of the testsubject is higher than the level of RNA encoded by the gene in blood ofhuman control subjects not having colorectal cancer classifies the testsubject as more likely to have colorectal cancer than to not havecolorectal cancer.

An embodiment of the methods of classifying a human test subject as morelikely to have colorectal cancer than to not have colorectal cancer ofthe invention includes determining of the level of RNA encoded by thegene in blood of the test subject is effected by determining the levelof RNA encoded by the gene in a blood sample isolated from the testsubject. An embodiment of the invention's methods of classifying a humantest subject as more likely to have colorectal cancer than to not havecolorectal cancer of the invention includes further determining levelsof RNA encoded by the gene in blood of a population of human subjectsnot having colorectal cancer, thereby providing the negative controldata representing the levels of RNA encoded by the gene in blood ofhuman control subjects not having colorectal cancer. An embodiment ofthe invention's methods of classifying a human test subject as morelikely to have colorectal cancer than to not have colorectal cancer ofthe invention includes where the level of RNA encoded by the gene inblood of the test subject is determined via quantitative reversetranscriptase-polymerase chain reaction analysis. An embodiment of theinvention's methods of classifying a human test subject as more likelyto have colorectal cancer than to not have colorectal cancer of theinvention includes where the level of RNA encoded by the gene in bloodof the test subject and the levels of RNA encoded by the gene in bloodof the control subjects are determined via the same method. Anembodiment of the invention's methods of classifying a human testsubject as more likely to have colorectal cancer than to not havecolorectal cancer of the invention includes where the level of RNAencoded by the gene in blood of the test subject is determined as aratio to a level of RNA encoded by ACTB in blood of the test subject. Inan aspect of this embodiment, the level of RNA encoded by the gene inblood of the test subject and the level of RNA encoded by ACTB in bloodof the test subject are determined via duplex quantitative reversetranscriptase-polymerase chain reaction analysis of RNA encoded by thegene and of RNA encoded by ACTB. An embodiment of the invention'smethods of classifying a human test subject as more likely to havecolorectal cancer than to not have colorectal cancer includes where thelevel of RNA encoded by the gene in blood of the test subject isdetermined as a ratio to a level of RNA encoded by IL2RB in blood of thetest subject, and/or where the level of RNA encoded by the gene in bloodof the test subject and the level of RNA encoded by IL2RB in blood ofthe test subject are determined via duplex quantitative reversetranscriptase-polymerase chain reaction analysis of RNA encoded by thegene and of RNA encoded by IL2RB.

Another aspect of the invention disclosed herein is a computer-basedmethod of classifying a human test subject as more likely to havecolorectal cancer than to not have colorectal cancer, the methodcomprising computer-implemented steps of: (a) applying to test datarepresenting a level of RNA encoded by a ANXA3 gene in blood of the testsubject and to negative control data representing a level of RNA encodedby the gene in blood of human control subjects not having colorectalcancer a mathematical formula for generating a value indicating whetherthe level of RNA encoded by the gene in blood of the test subject ishigher than the level of RNA encoded by the gene in blood of humancontrol subjects not having colorectal cancer; and (b) outputting thevalue, where an indication by the value that the level of RNA encoded bythe gene in blood of the test subject is higher than the level of RNAencoded by the gene in blood of human control subjects not havingcolorectal cancer classifies the test subject as more likely to havecolorectal cancer than to not have colorectal cancer. Another aspect ofthe invention disclosed herein is a computer-based method of classifyinga human test subject as more likely to have colorectal cancer than tonot have colorectal cancer, the method comprising computer-implementedsteps of: (a) applying to test data representing a level of RNA encodedby a CLEC4D gene in blood of the test subject and to negative controldata representing a level of RNA encoded by the gene in blood of humancontrol subjects not having colorectal cancer a mathematical formula forgenerating a value indicating whether the level of RNA encoded by thegene in blood of the test subject is higher than the level of RNAencoded by the gene in blood of human control subjects not havingcolorectal cancer; and (b) outputting the value, where an indication bythe value that the level of RNA encoded by the gene in blood of the testsubject is higher than the level of RNA encoded by the gene in blood ofhuman control subjects not having colorectal cancer classifies the testsubject as more likely to have colorectal cancer than to not havecolorectal cancer. Another aspect of the invention disclosed herein is acomputer-based method of classifying a human test subject as more likelyto have colorectal cancer than to not have colorectal cancer, the methodcomprising computer-implemented steps of: (a) applying to test datarepresenting a level of RNA encoded by a IL2RB gene in blood of the testsubject and to negative control data representing a level of RNA encodedby the gene in blood of human control subjects not having colorectalcancer a mathematical formula for generating a value indicating whetherthe level of RNA encoded by the gene in blood of the test subject ishigher than the level of RNA encoded by the gene in blood of humancontrol subjects not having colorectal cancer; and (b) outputting thevalue, where an indication by the value that the level of RNA encoded bythe gene in blood of the test subject is lower than the level of RNAencoded by the gene in blood of human control subjects not havingcolorectal cancer classifies the test subject as more likely to havecolorectal cancer than to not have colorectal cancer. Another aspect ofthe invention disclosed herein is a computer-based method of classifyinga human test subject as more likely to have colorectal cancer than tonot have colorectal cancer, the method comprising computer-implementedsteps of: (a) applying to test data representing a level of RNA encodedby a LMNB1 gene in blood of the test subject and to negative controldata representing a level of RNA encoded by the gene in blood of humancontrol subjects not having colorectal cancer a mathematical formula forgenerating a value indicating whether the level of RNA encoded by thegene in blood of the test subject is higher than the level of RNAencoded by the gene in blood of human control subjects not havingcolorectal cancer; and (b) outputting the value, where an indication bythe value that the level of RNA encoded by the gene in blood of the testsubject is higher than the level of RNA encoded by the gene in blood ofhuman control subjects not having colorectal cancer classifies the testsubject as more likely to have colorectal cancer than to not havecolorectal cancer. Another aspect of the invention disclosed herein is acomputer-based method of classifying a human test subject as more likelyto have colorectal cancer than to not have colorectal cancer, the methodcomprising computer-implemented steps of: (a) applying to test datarepresenting a level of RNA encoded by a PRRG4 gene in blood of the testsubject and to negative control data representing a level of RNA encodedby the gene in blood of human control subjects not having colorectalcancer a mathematical formula for generating a value indicating whetherthe level of RNA encoded by the gene in blood of the test subject ishigher than the level of RNA encoded by the gene in blood of humancontrol subjects not having colorectal cancer; and (b) outputting thevalue, where an indication by the value that the level of RNA encoded bythe gene in blood of the test subject is lower than the level of RNAencoded by the gene in blood of human control subjects not havingcolorectal cancer classifies the test subject as more likely to havecolorectal cancer than to not have colorectal cancer. Another aspect ofthe invention disclosed herein is a computer-based method of classifyinga human test subject as more likely to have colorectal cancer than tonot have colorectal cancer, the method comprising computer-implementedsteps of: (a) applying to test data representing a level of RNA encodedby a TNFAIP6 gene in blood of the test subject and to negative controldata representing a level of RNA encoded by the gene in blood of humancontrol subjects not having colorectal cancer a mathematical formula forgenerating a value indicating whether the level of RNA encoded by thegene in blood of the test subject is higher than the level of RNAencoded by the gene in blood of human control subjects not havingcolorectal cancer; and (b) outputting the value, where an indication bythe value that the level of RNA encoded by the gene in blood of the testsubject is lower than the level of RNA encoded by the gene in blood ofhuman control subjects not having colorectal cancer classifies the testsubject as more likely to have colorectal cancer than to not havecolorectal cancer. Another aspect of the invention disclosed herein is acomputer-based method of classifying a human test subject as more likelyto have colorectal cancer than to not have colorectal cancer, the methodcomprising computer-implemented steps of: (a) applying to test datarepresenting a level of RNA encoded by a VNN1 gene in blood of the testsubject and to negative control data representing a level of RNA encodedby the gene in blood of human control subjects not having colorectalcancer a mathematical formula for generating a value indicating whetherthe level of RNA encoded by the gene in blood of the test subject ishigher than the level of RNA encoded by the gene in blood of humancontrol subjects not having colorectal cancer; and (b) outputting thevalue, where an indication by the value that the level of RNA encoded bythe gene in blood of the test subject is lower than the level of RNAencoded by the gene in blood of human control subjects not havingcolorectal cancer classifies the test subject as more likely to havecolorectal cancer than to not have colorectal cancer.

An embodiment of the invention's computer-based methods of classifying ahuman test subject as more likely to have colorectal cancer than to nothave colorectal cancer, includes where the level of RNA encoded by thegene in blood of the test subject is determined via quantitative reversetranscriptase-polymerase chain reaction analysis. An embodiment of theinvention's computer-based methods of classifying a human test subjectas more likely to have colorectal cancer than to not have colorectalcancer, includes where the level of RNA encoded by the gene in blood ofthe test subject and the levels of RNA encoded by the gene in blood ofthe control subjects are determined via the same method. An embodimentof the invention's computer-based methods of classifying a human testsubject as more likely to have colorectal cancer than to not havecolorectal cancer, includes where the level of RNA encoded by the genein blood of the test subject is determined as a ratio to a level of RNAencoded by ACTB in blood of the test subject. An aspect of thisembodiment includes where the level of RNA encoded by the gene in bloodof the test subject and the level of RNA encoded by ACTB in blood of thetest subject are determined via duplex quantitative reversetranscriptase-polymerase chain reaction analysis of RNA encoded by thegene and of RNA encoded by ACTB. An embodiment of the invention'scomputer-based methods of classifying a human test subject as morelikely to have colorectal cancer than to not have colorectal cancer,includes where the level of RNA encoded by the gene in blood of the testsubject is determined as a ratio to a level of RNA encoded by IL2RB inblood of the test subject. An aspect of this embodiment includes wherethe level of RNA encoded by the gene in blood of the test subject andthe level of RNA encoded by IL2RB in blood of the test subject aredetermined via duplex quantitative reverse transcriptase-polymerasechain reaction analysis of RNA encoded by the gene and of RNA encoded byIL2RB.

Another aspect of the invention disclosed herein is a method ofclassifying a human test subject as more likely to have colorectalcancer than to not have colorectal cancer, the method comprising, foreach gene of a set of one or more genes selected from the groupconsisting of ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1: (a)determining a level of RNA encoded by the gene in blood of the testsubject, thereby generating test data; (b) providing negative controldata representing levels of RNA encoded by the gene in blood of humancontrol subjects not having colorectal cancer; and (c) applying to thetest data and to the negative control data a mathematical formula forgenerating a value indicating, for ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6and VNN1, whether the level of RNA encoded by the gene in blood of thetest subject is higher than the level of RNA encoded by the gene inblood of human control subjects not having colorectal cancer, andindicating, for IL2RB, whether the level of RNA encoded by the gene inblood of the test subject is lower than the level of RNA encoded by thegene in blood of human control subjects not having colorectal cancer,where, for ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1, an indicationby the value that the level of RNA encoded by the gene in blood of thetest subject is higher than the level of RNA encoded by the gene inblood of human control subjects not having colorectal cancer classifiesthe test subject as more likely to have colorectal cancer than to nothave colorectal cancer, and where, for IL2RB, an indication by the valuethat the level of RNA encoded by the gene in blood of the test subjectis lower than the level of RNA encoded by the gene in blood of humancontrol subjects not having colorectal cancer classifies the testsubject as more likely to have colorectal cancer than to not havecolorectal cancer. An embodiment of this aspect includes determining ofthe level of RNA encoded by the gene in blood of the test subject iseffected by determining the level of RNA encoded by the gene in a bloodsample isolated from the test subject. Another embodiment of this aspectincludes further comprising determining levels of RNA encoded by thegene in blood of a population of human subjects having colorectalcancer, thereby providing the positive control data representing thelevels of RNA encoded by the gene in blood of human control subjectshaving colorectal cancer, and determining levels of RNA encoded by thegene in blood of a population of human subjects not having colorectalcancer, thereby providing the negative control data representing thelevels of RNA encoded by the gene in blood of human control subjects nothaving colorectal cancer. Anther embodiment of this aspect includeswhere the level of RNA encoded by the gene in blood of the test subjectis determined via quantitative reverse transcriptase-polymerase chainreaction analysis. An embodiment of this aspect includes where the levelof RNA encoded by the gene in blood of the test subject and the levelsof RNA encoded by the gene in blood of the control subjects aredetermined via the same method. An embodiment of this aspect includeswhere the level of RNA encoded by the gene in blood of the test subjectis determined as a ratio to a level of RNA encoded by ACTB in blood ofthe test subject. An further embodiment includes where the level of RNAencoded by the gene in blood of the test subject and the level of RNAencoded by ACTB in blood of the test subject are determined via duplexquantitative reverse transcriptase-polymerase chain reaction analysis ofRNA encoded by the gene and of RNA encoded by ACTB. An embodiment ofthis aspect includes where the set of one or more genes is a set of oneor more genes selected from the group consisting of ANXA3, CLEC4D,LMNB1, PRRG4, TNFAIP6 and VNN1, and where the level of RNA encoded bythe gene in blood of the test subject is determined as a ratio to alevel of RNA encoded by IL2RB in blood of the test subject. Anembodiment of this aspect includes where the level of RNA encoded by thegene in blood of the test subject and the level of RNA encoded by IL2RBin blood of the test subject are determined via duplex quantitativereverse transcriptase-polymerase chain reaction analysis of RNA encodedby the gene and of RNA encoded by IL2RB.

Another aspect of the invention disclosed herein is a computer-basedmethod of classifying a human test subject as more likely to havecolorectal cancer than to not have colorectal cancer, the methodcomprising, for each gene of a set of one or more genes selected fromthe group consisting of ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 andVNN1, computer-implemented steps of: (a) applying to test datarepresenting a level of RNA encoded by the gene in blood of the testsubject and to negative control data representing a level of RNA encodedby the gene in blood of human control subjects not having colorectalcancer, a formula for calculating a value indicating, for ANXA3, CLEC4D,LMNB1, PRRG4, TNFAIP6 and VNN1, whether the level of RNA encoded by thegene in blood of the test subject is higher than the level of RNAencoded by the gene in blood of human control subjects not havingcolorectal cancer, and indicating, for IL2RB, whether the level of RNAencoded by the gene in blood of the test subject is lower than the levelof RNA encoded by the gene in blood of human control subjects not havingcolorectal cancer, where, for ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 andVNN1, an indication that the level of RNA encoded by the gene in bloodof the test subject is higher than the level of RNA encoded by the genein blood of human control subjects not having colorectal cancerclassifies the test subject as more likely to have colorectal cancerthan to not have colorectal cancer, and where, for IL2RB, an indicationthat the level of RNA encoded by the gene in blood of the test subjectis lower than the level of RNA encoded by the gene in blood of humancontrol subjects not having colorectal cancer classifies the testsubject as more likely to have colorectal cancer than to not havecolorectal cancer. An embodiment of this aspect of the inventiondisclosed herein includes where the level of RNA encoded by the gene inblood of the test subject is determined via quantitative reversetranscriptase-polymerase chain reaction analysis. Another embodiment ofthis aspect of the invention disclosed herein includes where the levelof RNA encoded by the gene in blood of the test subject and the levelsof RNA encoded by the gene in blood of the control subjects aredetermined via the same method. Another embodiment of this aspect of theinvention disclosed herein includes where the level of RNA encoded bythe gene in blood of the test subject is determined as a ratio to alevel of RNA encoded by ACTB in blood of the test subject. In a furtherembodiment of this embodiment of the invention as disclosed herein iswhere the level of RNA encoded by the gene in blood of the test subjectand the level of RNA encoded by ACTB in blood of the test subject aredetermined via duplex quantitative reverse transcriptase-polymerasechain reaction analysis of RNA encoded by the gene and of RNA encoded byACTB. Another embodiment of this aspect of the invention disclosedherein includes where the set of one or more genes is a set of one ormore genes selected from the group consisting of ANXA3, CLEC4D, LMNB1,PRRG4, TNFAIP6 and VNN1, and where the level of RNA encoded by the genein blood of the test subject is determined as a ratio to a level of RNAencoded by IL2RB in blood of the test subject. In a further embodimentof this embodiment of the invention as disclosed herein, the level ofRNA encoded by the gene in blood of the test subject and the level ofRNA encoded by IL2RB in blood of the test subject are determined viaduplex quantitative reverse transcriptase-polymerase chain reactionanalysis of RNA encoded by the gene and of RNA encoded by IL2RB. In afurther embodiment of this embodiment of the invention as disclosedherein, the set of one or more genes consists of PRRG4. In a furtherembodiment of this embodiment of the invention as disclosed herein, theset of one or more genes consists of IL2RB and PRRG4.

Another aspect of the invention disclosed herein is a kit comprisingpackaging and containing, for each gene of a set of two or more genesselected from the group consisting of ACTB, ANXA3, CLEC4D, IL2RB, LMNB1,PRRG4, TNFAIP6 and VNN1, a primer set capable of generating anamplification product of DNA complementary to RNA encoded, in a humansubject, only by the gene. In an embodiment of this aspect of theinvention disclosed herein, the kit further containing two or morecomponents selected from the group consisting of a thermostablepolymerase, a reverse transcriptase, deoxynucleotide triphosphates,nucleotide triphosphates and enzyme buffer. In another embodiment ofthis aspect of the invention disclosed herein, the kit further containsat least one labelled probe capable of selectively hybridizing to eithera sense or an antisense strand of the amplification product. In anotherembodiment of this aspect of the invention disclosed herein, the set ofone or more genes of the kit consists of ACTB and one or more genesselected from the group consisting of ANXA3, CLEC4D, IL2RB, LMNB1,PRRG4, TNFAIP6 and VNN1. In another embodiment of this aspect of theinvention disclosed herein, the set of one or more genes of the kitconsists of ACTB and ANXA3. In another embodiment of this aspect of theinvention disclosed herein, the set of one or more genes of the kitconsists of ACTB and CLEC4D. In another embodiment of this aspect of theinvention disclosed herein, the set of one or more genes of the kitconsists of ACTB and IL2RB. In another embodiment of this aspect of theinvention disclosed herein, the set of one or more genes of the kitconsists of ACTB and LMNB1. In another embodiment of this aspect of theinvention disclosed herein, the set of one or more genes of the kitconsists of ACTB and PRRG4. In another embodiment of this aspect of theinvention disclosed herein, the set of one or more genes of the kitconsists of ACTB and TNFAIP6. In another embodiment of this aspect ofthe invention disclosed herein, the set of one or more genes of the kitconsists of ACTB and VNN1. In another embodiment of this aspect of theinvention disclosed herein, the set of one or more genes of the kitconsists of IL2RB and one or more genes selected from the groupconsisting of ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1. In anotherembodiment of this aspect of the invention disclosed herein, the set ofone or more genes of the kit consists IL2RB and ANXA3. In anotherembodiment of this aspect of the invention disclosed herein, the set ofone or more genes of the kit consists of IL2RB and CLEC4D. In anotherembodiment of this aspect of the invention disclosed herein, the set ofone or more genes of the kit consists of IL2RB and LMNB1. In anotherembodiment of this aspect of the invention disclosed herein, the set ofone or more genes of the kit consists of IL2RB and PRRG4. In anotherembodiment of this aspect of the invention disclosed herein, the set ofone or more genes of the kit consists of IL2RB and TNFAIP6. In anotherembodiment of this aspect of the invention disclosed herein, the set ofone or more genes of the kit consists of IL2RB and VNN1.

Another aspect of the invention disclosed herein is a method ofdetermining whether a test subject is at an increased risk of havingcolorectal cancer relative to the general population, comprising: (a)obtaining a test sample of blood from the subject; and (i) determining alevel of RNA encoded by a annexin A3 (ANXA3) gene in the test sample ofblood, (ii) comparing the level of RNA encoded by ANXA3 as determined instep (i) with a level of the RNA encoded by the gene in control samplesof blood; and (b) concluding that the subject is at an increased risk ofhaving colorectal cancer relative to the general population if the levelof RNA encoded by the gene in the test sample of blood is higher than inthe control samples of blood. Another aspect of the invention disclosedherein is a method of determining whether a test subject is at anincreased risk of having colorectal cancer relative to the generalpopulation, comprising: (a) obtaining a test sample of blood from thesubject; and (i) determining a level of RNA encoded by a C-type lectindomain family 4, member D (CLEC4D) gene in the test sample of blood,(ii) comparing the level of RNA encoded by the gene as determined instep (i) with the level of the RNA encoded by the gene in controlsamples of blood; and (b) concluding that the subject is at an increasedrisk of having colorectal cancer relative to the general population ifthe level of RNA encoded by the gene in the test sample of blood ishigher than in the control samples of blood. Another aspect of theinvention disclosed herein is a method of determining whether a testsubject is at an increased risk of having colorectal cancer relative tothe general population, comprising: (a) obtaining a test sample of bloodfrom the subject; and (i) determining a level of RNA encoded by ainterleukin 2 receptor, beta (IL2RB) gene in the test sample of blood,(ii) comparing the level of RNA encoded by the gene as determined instep (i) with the level of the RNA encoded by the gene in controlsamples of blood; and (b) concluding that the subject is at an increasedrisk of having colorectal cancer relative to the general population ifthe level of RNA encoded by the gene in the test sample of blood islower than in the control samples of blood. Another aspect of theinvention disclosed herein is a method of determining whether a testsubject is at an increased risk of having colorectal cancer relative tothe general population, comprising: (a) obtaining a test sample of bloodfrom the subject; and (i) determining a level of RNA encoded by a laminB1 (LMNB1) gene in the test sample of blood, (ii) comparing the level ofRNA encoded by the gene as determined in step (i) with the level of theRNA encoded by the gene in control samples of blood; and (b) concludingthat the subject is at an increased risk of having colorectal cancerrelative to the general population if the level of RNA encoded by thegene in the test sample of blood is higher than in the control samplesof blood. Another aspect of the invention disclosed herein is a methodof determining whether a test subject is at an increased risk of havingcolorectal cancer relative to the general population, comprising: (a)obtaining a test sample of blood from the subject; and (i) determining alevel of RNA encoded by a proline rich Gla (G carboxyglutamic acid) 4(transmembrane) (PRRG4) gene in the test sample of blood, (ii) comparingthe level of RNA encoded by the gene as determined in step (i) with thelevel of the RNA encoded by the gene in control samples of blood; and(b) concluding that the subject is at an increased risk of havingcolorectal cancer relative to the general population if the level of RNAencoded by the gene in the test sample of blood is higher than in thecontrol samples of blood. Another aspect of the invention disclosedherein is a method of determining whether a test subject is at anincreased risk of having colorectal cancer relative to the generalpopulation, comprising: (a) obtaining a test sample of blood from thesubject; and (i) determining a level of RNA encoded by a tumor necrosisfactor, alpha induced protein 6 gene (TNFAIP6) in the test sample ofblood, (ii) comparing the level of RNA encoded by the gene as determinedin step (i) with the level of the RNA encoded by the gene in controlsamples of blood; and (b) concluding that the subject is at an increasedrisk of having colorectal cancer relative to the general population ifthe level of RNA encoded by the gene in the test sample of blood ishigher than in the control samples of blood. Another aspect of theinvention disclosed herein is a method of determining whether a testsubject is at an increased risk of having colorectal cancer relative tothe general population, comprising: (a) obtaining a test sample of bloodfrom the subject; and (i) determining a level of RNA encoded by a vanin1 (VNN1) gene in the test sample of blood, (ii) comparing the level ofRNA encoded by the gene as determined in step (i) with the level of theRNA encoded by the gene in control samples of blood; and (b) concludingthat the subject is at an increased risk of having colorectal cancerrelative to the general population if the level of RNA encoded by thegene in the test sample of blood is higher than in the control samplesof blood. In an embodiment of any one of these eight aspects thesemethods of determining whether a test subject is at an increased risk ofhaving colorectal cancer relative to the general population, the controlsamples are from individuals who have been diagnosed as not havingcolorectal cancer.

Another aspect of the invention disclosed herein is a method ofdetermining whether a test subject is at an increased risk of havingcolorectal cancer relative to the general population, comprising: (a)obtaining a test sample of blood from the subject; and for each gene ofa set of genes selected from the group consisting of: ANXA3, CLEC4D,IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1, (i) determining a level of RNAencoded by the gene in the test sample of blood, thereby generating testdata; and (ii) applying to the test data and to control datarepresenting a level of RNA encoded by the gene in one or more controlsamples of blood a mathematical formula for generating a valueindicating, for ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1, whetherthe level of RNA encoded by the gene in the test sample of blood ishigher than in the control samples of blood, and, for IL2RB, whether thelevel of RNA encoded by the gene in the test sample of blood is lowerthan in the control samples of blood; and (b) concluding that thesubject is at an increased risk of having colorectal cancer relative tothe general population if, for ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 andVNN1, the value indicates that the level of RNA encoded by the gene inthe test sample of blood is higher than in the control samples of blood,and concluding that the subject is at an increased risk of havingcolorectal cancer relative to the general population if, for IL2RB, thevalue indicates that the level of RNA encoded by the gene in the testsample of blood is lower than in the control samples of blood. Anotheraspect of the invention disclosed herein is an isolated compositioncomprising a blood sample from a test subject and a nucleic acidmolecule selected from one or more of the group consisting of RNAencoded by an ANXA3 gene, cDNA complementary to the RNA, anoligonucleotide which specifically hybridizes to the cDNA or the RNAunder stringent conditions, a primer set capable of generating anamplification product of the cDNA complementary to RNA, and anamplification product of the cDNA. One embodiment of this compositionfurther comprises a nucleic acid molecule selected from one or more ofthe group consisting of RNA encoded by one or more genes selected fromthe group of genes consisting of CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6and VNN1, cDNA complementary to the RNA of the group of genes, anoligonucleotide which specifically hybridizes to the cDNA complementaryto the RNA of the group of genes or to the RNA of the group of genesunder stringent conditions, a primer set capable of generating anamplification product of the cDNA complementary to RNA of the group ofgenes, and an amplification product of the cDNA of the RNA of the groupof genes. Another aspect of the invention disclosed herein is anisolated composition comprising a blood sample from a test subject and anucleic acid molecule selected from one or more of the group consistingof RNA encoded by a CLEC4D gene, cDNA complementary to the RNA, anoligonucleotide which specifically hybridizes to the cDNA or the RNAunder stringent conditions, a primer set capable of generating anamplification product of the cDNA complementary to RNA, and anamplification product of the cDNA. One embodiment of this compositionfurther comprises a nucleic acid molecule selected from one or more ofthe group consisting of RNA encoded by one or more genes selected fromthe group of genes consisting of ANXA3, IL2RB, LMNB1, PRRG4, TNFAIP6 andVNN1, cDNA complementary to the RNA of the group of genes, anoligonucleotide which specifically hybridizes to the cDNA complementaryto the RNA of the group of genes or to the RNA of the group of genesunder stringent conditions, a primer set capable of generating anamplification product of the cDNA complementary to RNA of the group ofgenes, and an amplification product of the cDNA of the RNA of the groupof genes. Another aspect of the invention disclosed herein is anisolated composition comprising a blood sample from a test subject and anucleic acid molecule selected from one or more of the group consistingof RNA encoded by a IL2RB gene, cDNA complementary to the RNA, anoligonucleotide which specifically hybridizes to the cDNA or the RNAunder stringent conditions, a primer set capable of generating anamplification product of the cDNA complementary to RNA, and anamplification product of the cDNA. One embodiment of this compositionfurther comprises a nucleic acid molecule selected from one or more ofthe group consisting of RNA encoded by one or more genes selected fromthe group of genes consisting of ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6and VNN1, cDNA complementary to the RNA of the group of genes, anoligonucleotide which specifically hybridizes to the cDNA complementaryto the RNA of the group of genes or to the RNA of the group of genesunder stringent conditions, a primer set capable of generating anamplification product of the cDNA complementary to RNA of the group ofgenes, and an amplification product of the cDNA of the RNA of the groupof genes. Another aspect of the invention disclosed herein is anisolated composition comprising a blood sample from a test subject and anucleic acid molecule selected from one or more of the group consistingof RNA encoded by a LMNB1 gene, cDNA complementary to the RNA, anoligonucleotide which specifically hybridizes to the cDNA or the RNAunder stringent conditions, a primer set capable of generating anamplification product of the cDNA complementary to RNA, and anamplification product of the cDNA. One embodiment of this compositionfurther comprises a nucleic acid molecule selected from one or more ofthe group consisting of RNA encoded by one or more genes selected fromthe group of genes consisting of ANXA3, CLEC4D, IL2RB, PRRG4, TNFAIP6and VNN1, cDNA complementary to the RNA of the group of genes, anoligonucleotide which specifically hybridizes to the cDNA complementaryto the RNA of the group of genes or to the RNA of the group of genesunder stringent conditions, a primer set capable of generating anamplification product of the cDNA complementary to RNA of the group ofgenes, and an amplification product of the cDNA of the RNA of the groupof genes. Another aspect of the invention disclosed herein is anisolated composition comprising a blood sample from a test subject and anucleic acid molecule selected from one or more of the group consistingof RNA encoded by a PRRG4 gene, cDNA complementary to the RNA, anoligonucleotide which specifically hybridizes to the cDNA or the RNAunder stringent conditions, a primer set capable of generating anamplification product of the cDNA complementary to RNA, and anamplification product of the cDNA. One embodiment of this compositionfurther comprises nucleic acid molecule selected from one or more of thegroup consisting of RNA encoded by one or more genes selected from thegroup of genes consisting of ANXA3, CLEC4D, IL2RB, LMNB1, TNFAIP6 andVNN1, cDNA complementary to the RNA of the group of genes, anoligonucleotide which specifically hybridizes to the cDNA complementaryto the RNA of the group of genes or to the RNA of the group of genesunder stringent conditions, a primer set capable of generating anamplification product of the cDNA complementary to RNA of the group ofgenes, and an amplification product of the cDNA of the RNA of the groupof genes. Another aspect of the invention disclosed herein is anisolated composition comprising a blood sample from a test subject and anucleic acid molecule selected from one or more of the group consistingof RNA encoded by a TNFAIP6 gene, cDNA complementary to the RNA, anoligonucleotide which specifically hybridizes to the cDNA or the RNAunder stringent conditions, a primer set capable of generating anamplification product of the cDNA complementary to RNA, and anamplification product of the cDNA. One embodiment of this compositionfurther comprises a nucleic acid molecule selected from one or more ofthe group consisting of RNA encoded by one or more genes selected fromthe group of genes consisting of ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, andVNN1, cDNA complementary to the RNA of the group of genes, anoligonucleotide which specifically hybridizes to the cDNA complementaryto the RNA of the group of genes or to the RNA of the group of genesunder stringent conditions, a primer set capable of generating anamplification product of the cDNA complementary to RNA of the group ofgenes, and an amplification product of the cDNA of the RNA of the groupof genes. Another aspect of the invention disclosed herein is anisolated composition comprising a blood sample from a test subject and anucleic acid molecule selected from one or more of the group consistingof RNA encoded by a VNN1 gene, cDNA complementary to the RNA, anoligonucleotide which specifically hybridizes to the cDNA or the RNAunder stringent conditions, a primer set capable of generating anamplification product of the cDNA complementary to RNA, and anamplification product of the cDNA. One embodiment of this compositionfurther comprises a nucleic acid molecule selected from one or more ofthe group consisting of RNA encoded by one or more genes selected fromthe group of genes consisting of ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, andTNFAIP6, cDNA complementary to the RNA of the group of genes, anoligonucleotide which specifically hybridizes to the cDNA complementaryto the RNA of the group of genes or to the RNA of the group of genesunder stringent conditions, a primer set capable of generating anamplification product of the cDNA complementary to RNA of the group ofgenes, and an amplification product of the cDNA of the RNA of the groupof genes.

Another aspect of the invention disclosed herein is an isolatedcomposition comprising an isolated nucleic acid molecule of a bloodsample from a test subject, where the nucleic acid molecule is selectedfrom one or more of the group consisting of RNA encoded by an ANXA3gene, cDNA complementary to the RNA, an oligonucleotide whichspecifically hybridizes to the cDNA or the RNA under stringentconditions, a primer set capable of generating an amplification productof the cDNA complementary to RNA, and an amplification product of thecDNA. One embodiment of this composition further comprises a nucleicacid molecule selected from one or more of the group consisting of RNAencoded by one or more genes selected from the group of genes consistingof CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1, cDNA complementary tothe RNA of the group of genes or the complement thereof, anoligonucleotide which specifically hybridizes to the cDNA complementaryto the RNA of the group of genes or to the RNA of the group of genesunder stringent conditions, a primer set capable of generating anamplification product of the cDNA complementary to RNA of the group ofgenes, and an amplification product of the cDNA of the RNA of the groupof genes. Another aspect of the invention disclosed herein is anisolated composition comprising an isolated nucleic acid molecule of ablood sample from a test subject, where the nucleic acid molecule isselected from one or more of the group consisting of RNA encoded by anCLEC4D gene, cDNA complementary to the RNA, an oligonucleotide whichspecifically hybridizes to the cDNA or the RNA under stringentconditions, a primer set capable of generating an amplification productof the cDNA complementary to RNA, and an amplification product of thecDNA. One embodiment of this composition further comprises a nucleicacid molecule selected from one or more of the group consisting of RNAencoded by one or more genes selected from the group of genes consistingof ANXA3, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1, cDNA complementary tothe RNA of the group of genes or the complement thereof, anoligonucleotide which specifically hybridizes to the cDNA complementaryto the RNA of the group of genes or to the RNA of the group of genesunder stringent conditions, a primer set capable of generating anamplification product of the cDNA complementary to RNA of the group ofgenes, and an amplification product of the cDNA of the RNA of the groupof genes. Another aspect of the invention disclosed herein is anisolated composition comprising an isolated nucleic acid molecule of ablood sample from a test subject, where the nucleic acid molecule isselected from one or more of the group consisting of RNA encoded by anIL2RB gene, cDNA complementary to the RNA, an oligonucleotide whichspecifically hybridizes to the cDNA or the RNA under stringentconditions, a primer set capable of generating an amplification productof the cDNA complementary to RNA, and an amplification product of thecDNA. One embodiment of this composition further comprises a nucleicacid molecule selected from one or more of the group consisting of RNAencoded by one or more genes selected from the group of genes consistingof ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1, cDNA complementary tothe RNA of the group of genes or the complement thereof, anoligonucleotide which specifically hybridizes to the cDNA complementaryto the RNA of the group of genes or to the RNA of the group of genesunder stringent conditions, a primer set capable of generating anamplification product of the cDNA complementary to RNA of the group ofgenes, and an amplification product of the cDNA of the RNA of the groupof genes. Another aspect of the invention disclosed herein is anisolated composition comprising an isolated nucleic acid molecule of ablood sample from a test subject, where the nucleic acid molecule isselected from one or more of the group consisting of RNA encoded by anLMNB1 gene, cDNA complementary to the RNA, an oligonucleotide whichspecifically hybridizes to the cDNA or the RNA under stringentconditions, a primer set capable of generating an amplification productof the cDNA complementary to RNA, and an amplification product of thecDNA. One embodiment of this composition further comprises a nucleicacid molecule selected from one or more of the group consisting of RNAencoded by one or more genes selected from the group of genes consistingof ANXA3, CLEC4D, IL2RB, PRRG4, TNFAIP6 and VNN1, cDNA complementary tothe RNA of the group of genes or the complement thereof, anoligonucleotide which specifically hybridizes to the cDNA complementaryto the RNA of the group of genes or to the RNA of the group of genesunder stringent conditions, a primer set capable of generating anamplification product of the cDNA complementary to RNA of the group ofgenes, and an amplification product of the cDNA of the RNA of the groupof genes. Another aspect of the invention disclosed herein is anisolated composition comprising an isolated nucleic acid molecule of ablood sample from a test subject, where the nucleic acid molecule isselected from one or more of the group consisting of RNA encoded by aPRRG4, gene, cDNA complementary to the RNA, an oligonucleotide whichspecifically hybridizes to the cDNA or the RNA under stringentconditions, a primer set capable of generating an amplification productof the cDNA complementary to RNA, and an amplification product of thecDNA. One embodiment of this composition further comprises a nucleicacid molecule selected from one or more of the group consisting of RNAencoded by one or more genes selected from the group of genes consistingof ANXA3, CLEC4D, IL2RB, LMNB1, TNFAIP6 and VNN1, cDNA complementary tothe RNA of the group of genes or the complement thereof, anoligonucleotide which specifically hybridizes to the cDNA complementaryto the RNA of the group of genes or to the RNA of the group of genesunder stringent conditions, a primer set capable of generating anamplification product of the cDNA complementary to RNA of the group ofgenes, and an amplification product of the cDNA of the RNA of the groupof genes. Another aspect of the invention disclosed herein is anisolated composition comprising an isolated nucleic acid molecule of ablood sample from a test subject, where the nucleic acid molecule isselected from one or more of the group consisting of RNA encoded by aTNFAIP6 gene, cDNA complementary to the RNA, an oligonucleotide whichspecifically hybridizes to the cDNA or the RNA under stringentconditions, a primer set capable of generating an amplification productof the cDNA complementary to RNA, and an amplification product of thecDNA. One embodiment of this composition further comprises a nucleicacid molecule selected from one or more of the group consisting of RNAencoded by one or more genes selected from the group of genes consistingof ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, and VNN1, cDNA complementary tothe RNA of the group of genes or the complement thereof, anoligonucleotide which specifically hybridizes to the cDNA complementaryto the RNA of the group of genes or to the RNA of the group of genesunder stringent conditions, a primer set capable of generating anamplification product of the cDNA complementary to RNA of the group ofgenes, and an amplification product of the cDNA of the RNA of the groupof genes. An isolated composition comprising a blood sample from a testsubject and a nucleic acid molecule selected from one or more of thegroup consisting of RNA encoded by a VNN1 gene, cDNA complementary tothe RNA, an oligonucleotide which specifically hybridizes to the cDNA orthe RNA under stringent conditions, a primer set capable of generatingan amplification product of the cDNA complementary to RNA, and anamplification product of the cDNA. One embodiment of this compositionfurther comprises a nucleic acid molecule selected from one or more ofthe group consisting of RNA encoded by one or more genes selected fromthe group of genes consisting of ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, andTNFAIP6, cDNA complementary to the RNA of the group of genes or thecomplement thereof, an oligonucleotide which specifically hybridizes tothe cDNA complementary to the RNA of the group of genes or to the RNA ofthe group of genes under stringent conditions, a primer set capable ofgenerating an amplification product of the cDNA complementary to RNA ofthe group of genes, and an amplification product of the cDNA of the RNAof the group of genes.

Another aspect of the invention disclosed herein is a primer setcomprising a first primer, where the first primer is one of a set ofprimers capable of generating an amplification product of cDNAcomplementary to RNA of encoded by an ANXA3 gene, and a second primer,where the second primer is one of a set of primers capable of generatingan amplification product of cDNA complementary to RNA of encoded by aVNN1 gene, or composition thereof. Another aspect of the inventiondisclosed herein is a primer set comprising a first primer, where thefirst primer is one of a set of primers capable of generating anamplification product of cDNA complementary to RNA of encoded by anANXA3 gene, and a second primer, where the second primer is one of a setof primers capable of generating an amplification product of cDNAcomplementary to RNA of encoded by a TNFAIP6 gene, or compositionthereof. Another aspect of the invention disclosed herein is a primerset comprising a first primer, where the first primer is one of a set ofprimers capable of generating an amplification product of cDNAcomplementary to RNA of encoded by an ANXA3 gene, and a second primer,where the second primer is one of a set of primers capable of generatingan amplification product of cDNA complementary to RNA of encoded by aPRRG4 gene, or composition thereof. Another aspect of the inventiondisclosed herein is a primer set comprising a first primer, where thefirst primer is one of a set of primers capable of generating anamplification product of cDNA complementary to RNA of encoded by anANXA3 gene, and a second primer, where the second primer is one of a setof primers capable of generating an amplification product of cDNAcomplementary to RNA of encoded by a PRRG4 gene, or composition thereof.Another aspect of the invention disclosed herein is a primer setcomprising a first primer, where the first primer is one of a set ofprimers capable of generating an amplification product of cDNAcomplementary to RNA of encoded by an ANXA3 gene, and a second primer,where the second primer is one of a set of primers capable of generatingan amplification product of cDNA complementary to RNA of encoded by aLMNB1 gene, or composition thereof. Another aspect of the inventiondisclosed herein is a primer set comprising a first primer, where thefirst primer is one of a set of primers capable of generating anamplification product of cDNA complementary to RNA of encoded by anANXA3 gene, and a second primer, where the second primer is one of a setof primers capable of generating an amplification product of cDNAcomplementary to RNA of encoded by an IL2RB gene, or compositionthereof. Another aspect of the invention disclosed herein is a primerset comprising a first primer, where the first primer is one of a set ofprimers capable of generating an amplification product of cDNAcomplementary to RNA of encoded by an ANXA3 gene, and a second primer,where the second primer is one of a set of primers capable of generatingan amplification product of cDNA complementary to RNA of encoded by aCLEC4D gene, or composition thereof. Another aspect of the inventiondisclosed herein is a primer set comprising a first primer, where thefirst primer is one of a set of primers capable of generating anamplification product of cDNA complementary to RNA of encoded by anCLEC4D gene, and a second primer, where the second primer is one of aset of primers capable of generating an amplification product of cDNAcomplementary to RNA of encoded by a VNN1 gene, or composition thereof.Another aspect of the invention disclosed herein is a primer setcomprising a first primer, where the first primer is one of a set ofprimers capable of generating an amplification product of cDNAcomplementary to RNA of encoded by an CLEC4D gene, and a second primer,where the second primer is one of a set of primers capable of generatingan amplification product of cDNA complementary to RNA of encoded by aTNFAIP6 gene, or composition thereof. Another aspect of the inventiondisclosed herein is a primer set comprising a first primer, where thefirst primer is one of a set of primers capable of generating anamplification product of cDNA complementary to RNA of encoded by anCLEC4D gene, and a second primer, where the second primer is one of aset of primers capable of generating an amplification product of cDNAcomplementary to RNA of encoded by a PRRG4 gene, or composition thereof.Another aspect of the invention disclosed herein is a primer setcomprising a first primer, where the first primer is one of a set ofprimers capable of generating an amplification product of cDNAcomplementary to RNA of encoded by an CLEC4D gene, and a second primer,where the second primer is one of a set of primers capable of generatingan amplification product of cDNA complementary to RNA of encoded by aPRRG4 gene, or composition thereof. Another aspect of the inventiondisclosed herein is a primer set comprising a first primer, where thefirst primer is one of a set of primers capable of generating anamplification product of cDNA complementary to RNA of encoded by anCLEC4D gene, and a second primer, where the second primer is one of aset of primers capable of generating an amplification product of cDNAcomplementary to RNA of encoded by a LMNB1 gene, or composition thereof.Another aspect of the invention disclosed herein is a primer setcomprising a first primer, where the first primer is one of a set ofprimers capable of generating an amplification product of cDNAcomplementary to RNA of encoded by an CLEC4D gene, and a second primer,where the second primer is one of a set of primers capable of generatingan amplification product of cDNA complementary to RNA of encoded by anIL2RB gene, or composition thereof. Another aspect of the inventiondisclosed herein is a primer set comprising a first primer, where thefirst primer is one of a set of primers capable of generating anamplification product of cDNA complementary to RNA of encoded by anIL2RB gene, and a second primer, where the second primer is one of a setof primers capable of generating an amplification product of cDNAcomplementary to RNA of encoded by a TNFAIP6 gene, or compositionthereof. Another aspect of the invention disclosed herein is a primerset comprising a first primer, where the first primer is one of a set ofprimers capable of generating an amplification product of cDNAcomplementary to RNA of encoded by an IL2RB gene, and a second primer,where the second primer is one of a set of primers capable of generatingan amplification product of cDNA complementary to RNA of encoded by aPRRG4 gene, or composition thereof. Another aspect of the inventiondisclosed herein is a primer set comprising a first primer, where thefirst primer is one of a set of primers capable of generating anamplification product of cDNA complementary to RNA of encoded by anIL2RB gene, and a second primer, where the second primer is one of a setof primers capable of generating an amplification product of cDNAcomplementary to RNA of encoded by a LMNB1 gene, or composition thereof.Another aspect of the invention disclosed herein is a primer setcomprising a first primer, where the first primer is one of a set ofprimers capable of generating an amplification product of cDNAcomplementary to RNA of encoded by an IL2RB gene, and a second primer,where the second primer is one of a set of primers capable of generatingan amplification product of cDNA complementary to RNA of encoded by aVNN1 gene, or composition thereof. Another aspect of the inventiondisclosed herein is a primer set comprising a first primer, where thefirst primer is one of a set of primers capable of generating anamplification product of cDNA complementary to RNA of encoded by a LMNB1gene, and a second primer, where the second primer is one of a set ofprimers capable of generating an amplification product of cDNAcomplementary to RNA of encoded by a PRRG4 gene, or composition thereof.Another aspect of the invention disclosed herein is a primer setcomprising a first primer, where the first primer is one of a set ofprimers capable of generating an amplification product of cDNAcomplementary to RNA of encoded by a LMNB1 gene, and a second primer,where the second primer is one of a set of primers capable of generatingan amplification product of cDNA complementary to RNA of encoded by aTNFAIP6 gene, or composition thereof. Another aspect of the inventiondisclosed herein is a primer set comprising a first primer, where thefirst primer is one of a set of primers capable of generating anamplification product of cDNA complementary to RNA of encoded by anLMNB1 gene, and a second primer, where the second primer is one of a setof primers capable of generating an amplification product of cDNAcomplementary to RNA of encoded by a VNN1 gene, or composition thereof.Another aspect of the invention disclosed herein is a primer setcomprising a first primer, where the first primer is one of a set ofprimers capable of generating an amplification product of cDNAcomplementary to RNA of encoded by a PRRG4 gene, and a second primer,where the second primer is one of a set of primers capable of generatingan amplification product of cDNA complementary to RNA of encoded by aVNN1 gene, or composition thereof. Another aspect of the inventiondisclosed herein is a primer set comprising a first primer, where thefirst primer is one of a set of primers capable of generating anamplification product of cDNA complementary to RNA of encoded by a PRRG4gene, and a second primer, where the second primer is one of a set ofprimers capable of generating an amplification product of cDNAcomplementary to RNA of encoded by a TNFAIP6 gene, or compositionthereof. Another aspect of the invention disclosed herein is a primerset comprising a first primer, where the first primer is one of a set ofprimers capable of generating an amplification product of cDNAcomplementary to RNA of encoded by a VNN1 gene, and a second primer,where the second primer is one of a set of primers capable of generatingan amplification product of cDNA complementary to RNA of encoded by aTNFAIP6 gene, or composition thereof.

Another aspect of the invention disclosed herein is test systemcomprising: a) two or more blood samples where each blood sample is froma different test subject, and b) an isolated nucleic acid molecule ofeach the blood sample from a test subject, where the nucleic acidmolecule is selected from one or more of the group consisting of RNAencoded by an ANXA3 gene, cDNA complementary to the RNA, anoligonucleotide which specifically hybridizes to the cDNA or complementthereof, or the RNA under stringent conditions, a primer set capable ofgenerating an amplification product of the cDNA complementary to RNA,and an amplification product of the cDNA. Another aspect of theinvention disclosed herein is a test system comprising: a) two or moreblood samples where each blood sample is from a different test subject,and b) an isolated nucleic acid molecule of each the blood sample from atest subject, where the nucleic acid molecule is selected from one ormore of the group consisting of RNA encoded by a CLEC4D, gene, cDNAcomplementary to the RNA, an oligonucleotide which specificallyhybridizes to the cDNA or complement thereof, or the RNA under stringentconditions, a primer set capable of generating an amplification productof the cDNA complementary to RNA, and an amplification product of thecDNA Another aspect of the invention disclosed herein is a test systemcomprising: a) two or more blood samples where each blood sample is froma different test subject, and b) an isolated nucleic acid molecule ofeach the blood sample from a test subject, where the nucleic acidmolecule is selected from one or more of the group consisting of RNAencoded by an IL2RB gene, cDNA complementary to the RNA, anoligonucleotide which specifically hybridizes to the cDNA or complementthereof, or the RNA under stringent conditions, a primer set capable ofgenerating an amplification product of the cDNA complementary to RNA,and an amplification product of the cDNA. Another aspect of theinvention disclosed herein is a test system comprising: a) two or moreblood samples where each blood sample is from a different test subject,and b) an isolated nucleic acid molecule of each the blood sample from atest subject, where the nucleic acid molecule is selected from one ormore of the group consisting of RNA encoded by an LMNB1 gene, cDNAcomplementary to the RNA, an oligonucleotide which specificallyhybridizes to the cDNA or complement thereof, or the RNA under stringentconditions, a primer set capable of generating an amplification productof the cDNA complementary to RNA, and an amplification product of thecDNA. Another aspect of the invention disclosed herein is a test systemcomprising: a) two or more blood samples where each blood sample is froma different test subject, and b) an isolated nucleic acid molecule ofeach the blood sample from a test subject, where the nucleic acidmolecule is selected from one or more of the group consisting of RNAencoded by a PRRG4 gene, cDNA complementary to the RNA, anoligonucleotide which specifically hybridizes to the cDNA or complementthereof, or the RNA under stringent conditions, a primer set capable ofgenerating an amplification product of the cDNA complementary to RNA,and an amplification product of the cDNA. Another aspect of theinvention disclosed herein is a test system comprising: a) two or moreblood samples where each blood sample is from a different test subject,and b) an isolated nucleic acid molecule of each the blood sample from atest subject, where the nucleic acid molecule is selected from one ormore of the group consisting of RNA encoded by a TNFAIP6 gene, cDNAcomplementary to the RNA, an oligonucleotide which specificallyhybridizes to the cDNA or complement thereof, or the RNA under stringentconditions, a primer set capable of generating an amplification productof the cDNA complementary to RNA, and an amplification product of thecDNA. Another aspect of the invention disclosed herein is a test systemcomprising: a) two or more blood samples where each blood sample is froma different test subject, and b) an isolated nucleic acid molecule ofeach the blood sample from a test subject, where the nucleic acidmolecule is selected from one or more of the group consisting of RNAencoded by a VNN1 gene, cDNA complementary to the RNA, anoligonucleotide which specifically hybridizes to the cDNA or complementthereof, or the RNA under stringent conditions, a primer set capable ofgenerating an amplification product of the cDNA complementary to RNA,and an amplification product of the cDNA. An embodiment of any of thetest systems described in this paragraph includes where the test subjectis being screened for colorectal cancer.

The following non-limiting examples are illustrative of the invention:

EXAMPLES Example 1 General Materials and Methods

Introduction:

The following materials and methods describe experiments performed todemonstrate that analysis of blood for levels of RNA encoded by genessurprisingly identified by the present inventors as colorectal cancermarker genes in blood via array hybridization analysis using anAffymetrix U133Plus 2.0 GeneChip oligonucleotide array (Affymetrix;Santa Clara, Calif.) (data not shown), can also serve as blood markersfor diagnosing colorectal cancer via quantitative reverse-transcriptasePCR analysis.

Blood Sample Collection:

Samples of 2.5 ml whole blood were collected into PAXgene Blood RNATubes (PreAnalytiX) from human subjects not having any colorectalpathology and from human subjects having colorectal cancer. Samples wereobtained from subjects enrolled in colorectal cancer studies conductedby GeneNews Corp. and collaborating institutions. Blood samples fromsubjects having colorectal cancer were collected prior to tumorresection, and cancer stage and histology were determined byinstitutional pathologists. Blood samples from subjects not having anycolorectal pathology were collected from subjects presenting forendoscopy screening. Informed consent was obtained according to theresearch protocols approved by the research ethical boards of theinstitutions involved. Experimental group sample pairs were selectedwith an effort to match gender, age, body mass index (BMI), ethnicityand medical history. Samples were divided into training and test sets.

RNA Isolation:

A sample of 2.5 ml whole blood was collected into PAXgene Blood RNAtubes (PreAnalytiX) and processed in accordance with the instructions ofthe PAXgene Blood RNA Kit protocol. In brief, after storing the blood inthe PAXgene tube for at least 2 hours, the blood sample was centrifugedand the supernatant discarded. To the remaining sample, 350 microlitersof the supplied Buffer BR1 was added, and the sample was pipetted intothe spin column and centrifuged, washed and finally eluted as isolatedRNA and stored.

Reverse Transcription:

Reverse transcription of blood sample-derived RNA into single-strandedcomplementary DNA was performed using the High Capacity cDNA ReverseTranscription Kit from (Applied Biosystems; Foster City, Calif.; Productnumber 4368814), according to the manufacturer's instructions.Specifically, 1 microgram of isolated RNA was incubated with reversetranscriptase buffer, dNTPs, random primers and reverse transcriptaseand incubated at 25° C. for 10 minutes and subsequently at 37° C. fortwo hours.

Quantitative Real Time RT-PCR:

Quantitative real-time PCR analysis to measure levels of RNA encoded bythe genes listed in Table 1 was performed on cDNA samples using theQuantiTect™ Probe RT-PCR system (Qiagen; Valencia, Calif.; Product No.204345), using the primers listed in Table 2 for amplification of cDNAtemplate corresponding to the indicated gene, and TaqMan dual labeledprobes comprising the polynucleotides listed in Table 3 for measuringlevels of amplicon corresponding to the indicated gene. The TaqMan probeand primers were ordered from Applied Biosystems Assays-On-Demand, orfrom IDT (Integrated DNA Technologies, Coralville, Iowa), or fromBiosearch Technologies (Novato, Calif.). Amplicon levels were measuredin real time using a RealTime PCR System 7500 instrument (AppliedBiosystems). Specifically, 20 nanograms of cDNA resulting from reversetranscription was added to the QuantiTect Probe PCR Master Mix asprovided and no adjustments were made for magnesium concentration.Uracil-N-Glycosylase was not added. Both forward primer and reverseprimer (Table 1) specific to the target genes were added to aconcentration of 5 micromolar, and the resultant 25 microliter reactionvolume was incubated as follows: 50 degrees centigrade for 2 minutes,followed by 95 degrees centigrade for 15 minutes, followed by 40 cyclesof: [94 degrees centigrade for 15 seconds, followed by 55 degreescentigrade for 35 seconds, followed by 72 degrees centigrade for 30seconds]. Amplification data was collected during each of the 40incubations at 55 degrees centigrade. All quantitative reversetranscriptase-PCR analyses were performed as duplex amplifications of atarget gene and a reference gene (either ACTB or IL2RB, as indicated) inthe same reaction mixture. Serial dilution measurements for target andduplex partner genes were assayed, to ensure that the values were withinlinear range and that the amplification efficiencies were approximatelyequal. Examination via polyacrylamide gel electrophoresis providedconfirmation of specific PCR amplification and the lack of primer-dimerformation in each reaction well.

TABLE 1 Genes encoding target RNAs for determining colorectal cancerprobability versus absence of colorectal pathology. Gene GenBank SymbolAccession Gene Description ACTB NM_001101 beta-actin ANXA3 NM_005139annexin A3 CLEC4D NM_080387 C-type lectin domain family 4, member DIL2RB NM_000878 interleukin 2 receptor, beta LMNB1 NM_005573 lamin B1PRRG4 NM_024081 proline rich Gla (G-carboxyglutamic acid) 4(transmembrane) TNFAIP6 NM_007115 tumor necrosis factor, alpha-inducedprotein 6 VNN1 NM_004666 vanin 1

TABLE 2 Primers used for quantitative PCR analysis. Gene encodingamplified Primer Amplicon cDNA Primer Primer pair sequences positionsize (bp) ACTB 5′ 5′-CACCACACCTTCTACAATGAGCTG-3′ (SEQ ID NO: 1) 259 1583′ 5′-ACAGCCTGGATAGCAACGTACA-3′ (SEQ ID NO: 2) 416 ANXA3 5′5′-GAAACATCTGGTGACTTCCG-3′ (SEQ ID NO: 10) 748 103 3′5′-TCTGGGCATCTTGTTTGG-3 ′ (SEQ ID NO: 11) 850 CLEC4D 5′5′-CCATTTAACCCACGCAGAG-3′ (SEQ ID NO: 19) 673 101 3′5′-CAGGCCCATTTATCTTGGTT-3′ (SEQ ID NO: 20) 773 IL2RB 5′5′-AAATCTCCCAAGCCTCCCA-3′ (SEQ ID NO: 28) 588 127 3′5′-AGGCAGATCCATTCCTGCT-3′ (SEQ ID NO: 29) 714 LMNB1 5′5′-GGAGTGGTTGTTGAGGAAGAA-3′ (SEQ ID NO: 37) 2051 151 3′5′-CTGAGAAGGCTCTGCACTGTA-3′ (SEQ ID NO: 38) 2201 PRRG4 5′5′-ATGCGGGAGAAGAAGTGTTTAC-3′ (SEQ ID NO: 46) 341 153 3′5′-CTCTGGCTTCCTCATAATTGC-3′ (SEQ ID NO: 47) 493 TNFAIP6 5′5′-GCCTATTGCTACAACCCACA-3′ (SEQ ID NO: 55) 448 84 3′5′-TGGGAAGCCTGGAGATTTA-3′ (SEQ ID NO: 56) 531 VNN1 5′5′-TGACAGGAAGTGGCATCTAT-3′ (SEQ ID NO: 64) 835 147 3′5′-TACTGCTGGCATAGGAAGTC-3′ (SEQ ID NO: 65) 981

TABLE 3 TaqMan ® probes used for quantitative PCR analysis.Gene encoding Probe amplicon Taqman probe base sequence position ACTB5′-AACCGCGAGAAGATGACCCAGATCAT-3′ (SEQ ID NO: 3) 343 ANXA35′-TTGACTTTGGCAGATGGCAGA-3′ (SEQ ID NO: 12) 778 CLEC4D5′-CTGGCATAAGAATGAACCCGACA-3′ (SEQ ID NO: 21) 696 IL2RB5′-TTGAAAGACACCTGGAGTTCG-3 ′ (SEQ ID NO: 30) 612 LMNB15′-AACCCCAAGAGCATCCAATAG-3′ (SEQ ID NO: 39) 2089 PRRG45′-CTCTTCACTCCCGGCAACCTAGAA-3′ (SEQ ID NO: 48) 427 TNFAIP65′-AAGGAGTGTGGTGGCGTCTTTAC-3′ (SEQ ID NO: 57) 472 VNN15′-AGAAGAGGGAAAACTCCTCCTCTCG-3′ (SEQ ID NO: 66) 896

Determination of Observed Range of Fold-Changes in Levels of RNA Encodedby Marker Genes in Blood of Subjects Having Colorectal Cancer Relativeto Subjects not Having any Colorectal Pathology:

For each of the sample training and sample test sets, averagefold-change in levels of RNA encoded by marker genes, normalized toeither ACTB or IL2RB, were calculated as the ratio of average levels ofRNA encoded by marker genes in blood of subjects having colorectalcancer to average levels of RNA encoded by marker genes in blood ofsubjects not having any colorectal pathology. The statisticalsignificance of the fold-changes were confirmed by a p-value of lessthan 0.05. Maximum observed directional fold-changes in normalizedlevels of RNA encoded by marker genes found to be higher in blood ofsubjects having colorectal cancer than in blood of subjects not havingany colorectal pathology were further calculated, for each marker gene,as the ratio of the highest level observed in any single sample from asubject having colorectal cancer to the average level in subjects nothaving any colorectal pathology. Similarly, maximum observed directionalfold-changes in normalized levels of RNA encoded by marker genes foundto be lower in blood of subjects having colorectal cancer than in bloodof subjects not having any colorectal pathology were further calculated,for each marker gene, as the ratio of the lowest level observed in anysingle sample from a subject having colorectal cancer to the averagelevel in subjects not having any colorectal pathology. In this way,observed ranges of fold-changes, ranging from average fold-change tomaximal observed directional fold-change, in levels of RNA encoded bymarker genes in blood of subjects having colorectal cancer relative tosubjects not having any colorectal pathology were determined.

Formulation of Mathematical Models for Determining Probability ofColorectal Cancer Versus Absence of Colorectal Pathology:

Logistic regression was used to formulate mathematical models fordetermining the probability that a test subject has colorectal cancer asopposed to not having any colorectal pathology. Levels of RNA encoded bycolorectal cancer marker genes and of reference genes determined viaduplex quantitative reverse transcriptase PCR in blood of positive andnegative control subjects were analyzed via logistic regression so as togenerate models having the general form:

P={1+ê−[K ₀ +K ₁ L ₁ +K ₂ L ₂ +K ₃ L ₃ . . . +K _(n) L _(n)]}̂−1,

-   -   where P is the probability that a test subject has colorectal        cancer as opposed to not having any colorectal pathology; K₀ is        a constant; K₁ is a coefficient specific to a first marker gene;        L₁ is a ratio of a level of RNA encoded by the first gene to a        level of RNA encoded by a reference gene in blood of the test        subject; K₂ is a coefficient specific to a second marker gene;        L₂ is a ratio of a level of RNA encoded by the second gene to a        level of RNA encoded by the reference gene in blood of the test        subject; K₃ is a coefficient specific to a third marker gene; L₃        is a ratio of a level of RNA encoded by the third gene to a        level of RNA encoded by the reference gene in blood of the test        subject; K_(n) is a coefficient specific to an nth marker gene;        and L_(n) is a ratio of a level of RNA encoded by the nth gene        to a level of RNA encoded by the reference gene in blood of the        test subject. The ratio of the level of RNA encoded by a marker        gene to the level of RNA encoded by a reference gene was        calculated as the change (ΔCt) in the cycle number (Ct) at which        the increase in fluorescence is exponential between the marker        gene and the reference gene according to the equation: ΔCt=Ct        (marker gene)−Ct (reference gene). The caret symbol “̂” is used        herein to denote that a value preceding the caret is raised to a        power corresponding to the value following the caret.

Example 2 Measurement of Blood Levels of RNA Encoded by any Combinationof ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and/or VNN1 can be Usedto Determine a Probability that a Test Subject has Colorectal Cancer asOpposed to not Having any Colorectal Pathology

Materials and Methods:

Refer to “General materials and methods”, above.

Experimental Results:

Sample Training Set:

Discovery of Significantly Different Levels of RNA Encoded by ANXA3,CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1 and in Blood of SubjectsHaving Colorectal Cancer Relative to Subjects not Having any ColorectalPathology:

Quantitative reverse transcriptase PCR analysis of gene expression in atraining set of blood samples from 117 subjects having colorectal cancerand 130 subjects not having any colorectal pathology, using thehousekeeping gene ACTB as duplex partner for normalization of geneexpression levels was performed. The normalized RNA levels measured areshown in Table 4.

TABLE 4 Sample training set levels of RNA encoded by ANXA3, CLEC4D,IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1 in blood of subjects havingcolorectal cancer (Group 1) and subjects not having any colorectalpathology (Group 0), normalized to levels of RNA encoded by ACTB. Levelsshown correspond to ΔCt. Gene Sample ID Group ANXA3 CLEC4D IL2RB LMNB1PRRG4 TNFAIP6 VNN1 CD0011pax 0 6.523 7.755 5.195 7.310 7.860 8.501 8.991CD0012pax 0 7.878 8.595 5.250 7.525 8.183 8.439 9.878 CD0030pax 0 6.4116.420 4.173 6.220 6.973 5.901 7.101 CD0063pax 0 7.103 8.545 4.203 7.1657.795 8.499 8.628 CD0077pax 0 4.808 6.185 5.098 5.405 6.710 6.524 6.533CD0078pax 0 5.946 7.000 3.553 5.820 5.570 7.476 6.488 CD0085pax 0 5.5437.700 5.003 6.210 7.460 8.149 6.678 CD0117pax 0 6.021 8.170 4.463 5.6858.020 8.166 6.573 CD0146pax 0 5.396 6.335 4.468 5.320 5.735 6.691 6.253CD0167pax 0 3.501 4.893 4.480 4.978 5.590 6.469 5.173 CD0249pax 0 4.4434.855 4.878 4.803 6.043 5.556 6.471 CD0279pax 0 5.503 7.095 4.270 5.3956.098 6.694 7.043 CD0286pax 0 4.791 6.928 4.350 5.383 5.960 5.714 5.598CD0297pax 0 5.861 6.670 5.083 6.565 6.405 6.186 7.118 CD0323pax 0 6.9667.773 4.645 5.723 7.470 8.149 8.738 CD0445pax 0 6.458 7.420 4.448 6.1036.448 7.216 7.411 CD0463pax 0 4.391 6.485 4.203 5.605 6.583 6.161 7.036CD0491pax 0 5.093 6.370 4.928 6.123 6.978 7.171 6.511 CD0496pax 0 6.0588.270 4.670 6.355 7.783 6.434 6.623 CD0501pax 0 6.326 7.725 4.613 6.2707.215 8.581 6.978 CD0504pax 0 4.023 5.060 4.858 5.920 7.530 5.289 6.113CD0573pax 0 6.791 6.140 4.248 6.160 6.713 7.646 7.286 CD0578pax 0 6.3286.670 4.128 5.128 6.033 6.411 7.081 CD0620pax 0 2.361 3.628 6.120 3.8736.120 5.274 5.613 CD0639pax 0 5.611 7.013 4.980 6.258 5.630 6.689 7.528CD0645pax 0 4.596 5.868 5.190 4.908 5.475 6.099 5.608 CD0679pax 0 5.6117.808 5.070 5.633 6.150 7.974 7.748 CD0685pax 0 5.796 8.150 4.358 6.0507.155 7.606 6.673 CD0716pax 0 6.961 8.193 4.090 5.648 5.750 6.669 7.388CD0749pax 0 5.208 6.970 4.520 5.565 6.430 7.176 6.316 CD0760pax 0 2.8685.020 5.603 3.605 5.790 4.679 5.663 CD0811pax 0 7.188 8.065 3.275 6.5007.305 6.021 6.141 CD0846pax 0 4.626 6.488 4.730 5.308 5.645 4.864 7.813CD0848pax 0 5.113 6.235 3.380 5.915 6.388 6.409 7.263 CD0924pax 0 5.7316.370 5.238 6.095 6.248 5.791 6.131 CD1066pax 0 5.346 5.900 4.903 5.9906.868 7.466 6.211 CD1073pax 0 5.681 5.813 5.150 5.858 6.350 6.674 6.663CD1075pax 0 5.128 7.010 4.535 6.075 8.228 7.264 6.563 CD1089pax 0 5.0817.225 5.733 5.550 6.395 6.546 6.668 CD1116pax 0 4.188 5.500 5.023 5.2956.180 5.764 6.378 CD1120pax 0 3.663 5.495 5.203 4.780 6.555 6.864 5.118CD1198pax 0 5.398 6.210 4.155 6.055 6.095 6.086 5.881 PB1179pax 0 6.4017.293 4.700 6.563 7.080 8.169 7.418 PB1277pax 0 6.403 7.520 4.860 6.2607.185 7.586 6.351 PB1301pax 0 4.733 6.200 6.268 5.608 6.863 5.891 5.531PB1315pax 0 3.898 6.165 5.438 4.998 6.008 5.206 7.161 PB1345pax 0 5.2466.280 4.598 6.410 6.775 6.051 7.663 PB1518pax 0 6.806 7.803 5.055 5.8187.230 7.134 7.328 PB1520pax 0 4.283 5.560 3.250 4.660 3.793 5.214 6.108PB1574pax 0 7.538 8.685 5.583 7.453 7.483 7.166 8.226 PB1783pax 0 8.0569.095 5.453 7.650 8.535 9.011 8.448 PB1799pax 0 7.338 8.760 5.765 7.4358.855 9.181 7.801 PB1811pax 0 6.848 8.115 5.080 7.140 8.390 7.416 7.471PB1830pax 0 5.788 7.385 5.923 6.440 6.870 7.444 9.253 PB1833pax 0 5.9437.620 5.488 6.615 7.620 6.829 8.098 PB1843pax 0 7.218 7.870 5.780 6.7708.338 5.739 8.708 PB1851pax 0 7.468 8.045 6.050 7.795 8.325 10.181 9.261PB1919pax 0 8.271 9.525 5.233 7.165 8.975 9.051 9.108 PB1922pax 0 6.7888.280 6.028 7.363 8.348 9.281 8.306 PB1924pax 0 7.748 8.655 6.758 7.6288.303 8.816 9.401 PB1937pax 0 7.178 9.085 5.118 7.665 9.740 8.754 8.798PB1964pax 0 5.491 7.820 4.463 6.065 7.843 7.726 8.496 PB2027pax 0 5.4636.525 5.150 6.300 7.360 7.401 7.546 PB2029pax 0 5.793 7.060 5.050 6.3756.973 7.624 7.758 PB2073pax 0 6.021 6.335 4.873 6.290 7.933 6.991 6.846PB2086pax 0 5.048 5.665 4.208 5.098 6.453 5.286 5.991 PB2099pax 0 4.8085.360 5.388 5.910 6.680 5.604 7.003 PB2100pax 0 6.353 6.960 4.465 6.0857.625 7.351 6.826 PB2132pax 0 5.693 7.235 4.438 5.745 6.995 7.649 7.518PB2168pax 0 6.776 7.593 4.605 6.168 7.125 8.284 6.993 PB2192pax 0 6.7017.845 5.483 6.230 6.965 7.751 8.378 PB2196pax 0 6.061 7.655 4.573 6.1957.985 7.781 6.668 PB2200pax 0 6.706 7.640 5.158 6.025 7.103 6.826 6.276PB2213pax 0 6.898 7.435 4.773 6.380 6.630 8.614 7.448 PB2224pax 0 4.8415.385 4.743 5.210 6.448 7.546 6.851 PB2228pax 0 6.511 6.915 3.953 6.4507.373 8.906 8.086 PB2229pax 0 5.771 6.440 5.588 6.030 5.865 7.091 6.528PB2277pax 0 6.348 6.685 4.290 5.705 5.930 5.986 7.351 PB2297pax 0 5.8866.785 4.703 5.835 6.533 6.451 5.696 PB2312pax 0 5.533 6.530 3.773 6.0985.978 6.651 6.746 PB2398pax 0 4.711 5.390 5.033 5.440 5.748 5.136 7.111PB2409pax 0 5.946 7.195 4.933 5.835 6.950 6.811 7.683 PB2414pax 0 7.8437.790 3.955 6.380 7.745 9.026 7.101 PB2467pax 0 5.773 6.935 4.260 5.9556.525 7.596 6.756 PB2473pax 0 6.818 8.275 5.530 7.375 8.405 7.586 8.561PB2512pax 0 5.603 7.355 4.340 6.215 6.345 6.926 7.511 PB2568pax 0 5.3265.850 5.303 5.710 7.178 7.561 7.331 PB2571pax 0 5.561 5.995 4.523 6.0606.173 7.396 7.791 PB2603pax 0 5.778 6.480 3.953 5.903 6.313 7.021 5.986PB2624pax 0 5.383 5.465 3.948 5.498 6.838 6.146 6.741 PB2824pax 0 5.7816.748 4.675 5.758 6.830 7.154 7.223 PB2880pax 0 5.906 6.090 4.728 6.1605.218 7.361 7.386 PB3088pax 0 6.601 6.760 3.858 5.725 6.395 8.456 7.378RC0882pax 0 5.043 6.540 4.533 5.708 6.083 7.351 7.151 RC0888pax 0 4.7265.740 4.948 5.330 6.775 6.516 6.168 RC0968pax 0 3.238 3.590 4.008 4.3036.118 4.716 7.086 RC2114pax 0 4.473 5.900 4.768 5.168 6.028 5.216 7.446RC2238pax 0 7.318 8.785 5.878 7.175 8.665 9.209 9.903 RC2681pax 0 6.3317.515 5.623 6.995 7.833 6.471 7.536 RC2703pax 0 8.093 8.360 5.973 7.0538.253 7.801 8.091 RC2749pax 0 6.448 8.695 5.895 7.205 7.905 7.726 8.666RC2750pax 0 5.578 6.650 6.753 6.428 7.993 7.761 7.111 RC2756pax 0 7.4788.135 5.340 7.095 8.420 7.781 8.671 RC2771pax 0 5.848 8.345 6.240 6.4407.648 8.449 9.303 RC2790pax 0 8.086 9.228 6.880 7.573 8.475 8.679 8.948RC2792pax 0 7.956 8.058 5.850 7.068 8.320 9.219 8.448 RC2808pax 0 6.5568.790 5.233 6.605 7.815 7.096 7.653 RC2822pax 0 7.921 9.163 6.255 7.1938.075 10.284 7.718 RC2834pax 0 6.588 8.535 6.520 6.810 7.920 9.946 9.651RC2871pax 0 5.443 6.530 4.563 6.280 7.165 7.479 8.158 RC2879pax 0 6.2668.105 5.978 6.620 8.465 7.971 7.298 RC2892pax 0 6.086 7.423 5.185 6.1637.185 8.559 7.748 RC2895pax 0 6.148 6.900 4.378 6.270 7.450 6.109 7.473RC2921pax 0 6.846 7.623 4.720 6.758 7.520 7.954 8.073 RC2958pax 0 6.5816.735 4.863 6.185 6.638 5.851 7.616 RC3022pax 0 6.401 6.660 4.888 6.6857.925 7.776 6.578 RC3112pax 0 6.938 8.095 5.408 6.435 7.475 8.629 7.713RC3146pax 0 6.018 6.655 5.340 5.905 6.855 7.491 7.451 RC3184pax 0 6.9987.910 4.398 6.370 7.395 7.329 7.598 RC3232pax 0 5.021 6.460 5.003 5.0307.008 4.941 8.671 RC3324pax 0 5.158 6.220 5.203 5.055 6.935 6.869 5.713RC3327pax 0 5.238 5.225 4.708 5.313 5.253 6.381 7.541 RC3334pax 0 5.9537.670 4.710 6.135 6.850 6.816 8.081 RC3355pax 0 5.871 7.253 5.620 5.3586.325 7.699 6.648 RC3380pax 0 5.418 6.395 5.363 5.453 6.423 6.786 6.481RC3392pax 0 6.378 8.025 4.445 6.170 7.265 8.546 7.756 RC3413pax 0 6.1767.940 4.768 6.100 6.545 7.051 8.858 RC3421pax 0 5.661 5.515 5.088 5.9006.483 5.191 5.961 RC3468pax 0 5.831 5.890 5.443 5.905 6.523 7.171 6.636RC3498pax 0 5.553 5.515 5.593 6.145 7.200 7.484 6.468 CD0157pax 1 6.2236.530 4.198 6.115 6.930 7.654 8.168 CD0164pax 1 4.726 5.395 4.083 5.66010.733 7.791 6.696 CD0256pax 1 4.833 6.295 4.735 6.045 6.785 7.141 6.911CD0322pax 1 5.153 7.050 6.308 5.820 7.325 7.239 8.603 CD0356pax 1 5.2435.555 6.038 5.925 6.580 6.239 5.823 CD0371pax 1 5.643 7.110 5.073 6.0456.245 6.394 5.968 CD0629pax 1 4.453 5.995 4.503 5.555 7.380 5.114 6.563CD1050pax 1 6.238 5.930 4.943 6.150 7.105 6.969 7.243 MH0001pax 1 7.2668.375 5.103 7.770 8.568 9.266 8.706 MH0009pax 1 6.078 7.150 5.990 6.3257.185 6.131 6.426 MH0011pax 1 2.393 4.420 8.808 4.258 6.888 3.846 5.756MH0012pax 1 4.673 6.965 5.368 5.970 7.680 6.659 7.043 MH0014pax 1 6.2668.155 5.003 6.395 6.995 9.436 7.983 MH0016pax 1 5.408 6.770 6.225 6.0506.635 6.181 6.561 MH0017pax 1 6.071 8.290 5.323 6.710 6.750 8.231 8.433MH0018pax 1 6.856 7.175 5.093 6.250 7.358 8.451 6.791 MH0021pax 1 6.9486.675 5.263 5.483 6.398 8.236 8.111 MH0022pax 1 6.471 7.508 5.280 6.2287.030 7.344 7.548 MH0024pax 1 5.016 5.640 4.488 5.340 5.793 5.211 6.241MH0026pax 1 4.351 6.775 5.558 5.440 6.840 5.861 6.028 MH0028pax 1 6.1836.815 5.818 5.918 5.883 5.986 6.176 MH0029pax 1 5.388 6.360 5.015 6.2555.925 6.846 6.831 MH0035pax 1 6.111 8.575 4.708 6.645 7.460 7.051 7.638MH0037pax 1 5.441 7.063 5.375 5.578 6.325 7.089 7.948 MH0038pax 1 7.2067.463 5.020 6.748 7.635 8.089 8.113 MH0039pax 1 4.036 5.113 5.110 5.2985.110 5.394 6.383 MH0042pax 1 4.643 5.560 4.425 5.900 5.785 4.876 5.901MH0050pax 1 3.763 6.495 4.908 4.718 5.698 6.641 7.721 MH0051pax 1 4.9415.693 6.225 5.818 4.795 4.044 6.338 PB1829pax 1 7.363 9.380 6.678 7.0737.428 8.841 9.241 PB1842pax 1 7.483 8.295 6.188 7.488 7.173 8.786 8.011PB1872pax 1 7.051 8.525 6.318 7.410 7.175 8.486 7.533 PB2857pax 1 4.2686.600 4.810 5.300 6.470 5.266 6.976 RC2919pax 1 7.106 7.488 4.420 6.4638.350 8.399 9.318 RC3062pax 1 5.006 6.200 4.513 5.195 6.340 5.271 5.538RC3277pax 1 5.068 6.360 4.770 5.360 6.205 5.701 6.136 RC3297pax 1 5.7486.970 4.728 5.843 6.493 8.451 7.206 RC3445pax 1 5.503 6.560 5.290 6.2956.560 6.711 7.021 RC3467pax 1 6.893 8.640 3.945 6.670 7.040 8.251 7.771CC0003pax 1 4.281 5.963 5.500 4.608 5.205 7.009 4.853 DC0001pax 1 5.7135.580 5.378 5.868 6.408 6.201 6.541 DC0002pax 1 6.323 6.125 5.423 6.3806.410 7.374 7.273 DS0003pax 1 4.816 6.663 6.615 5.248 7.380 6.849 6.398FC0005pax 1 5.953 6.735 5.618 6.605 6.980 6.649 8.698 FC0011pax 1 6.4587.550 7.063 6.915 7.950 8.539 8.338 FC0012pax 1 3.868 6.850 7.138 5.2856.375 6.849 7.063 JGA0001pax 1 5.426 6.250 7.448 6.125 8.173 6.586 7.096JGA0008pax 1 5.448 7.600 6.018 6.793 7.403 7.366 7.841 JH0002pax 1 6.1087.335 5.633 6.725 6.950 7.484 7.128 JH0003pax 1 6.053 6.635 5.090 5.9056.145 5.701 7.081 JH0004pax 1 5.373 5.985 5.265 5.935 6.570 7.371 6.961JH0005pax 1 5.341 6.565 5.008 5.780 6.688 7.356 7.091 JH0006pax 1 4.7715.840 5.073 5.525 6.198 6.731 5.551 JH0007pax 1 2.956 3.035 5.353 4.8156.483 5.661 4.916 JH0008pax 1 5.876 8.435 5.173 6.265 7.573 7.811 6.711JH0009pax 1 4.101 3.770 4.793 5.540 5.293 4.636 5.876 JH0010pax 1 5.0265.810 4.958 6.105 7.018 6.046 5.526 JH0012pax 1 4.981 5.435 4.718 5.9656.318 5.801 6.511 JH0013pax 1 5.501 6.610 5.268 5.905 7.048 8.861 7.191JH0014pax 1 5.053 5.235 4.253 4.735 4.860 6.259 7.488 JH0016pax 1 5.5966.390 4.438 5.980 6.338 5.566 6.191 JH0018pax 1 4.401 5.770 5.248 5.7656.348 5.696 6.446 JH0019pax 1 5.751 6.775 5.468 6.055 6.693 5.926 6.656JH0020pax 1 5.001 7.450 4.563 5.875 5.618 5.756 6.706 JH0021pax 1 5.7267.650 6.058 5.730 6.105 6.611 6.808 JH0023pax 1 4.696 6.805 3.873 5.0206.960 5.951 6.968 JH0024pax 1 6.008 7.895 5.113 6.865 7.620 9.099 8.698JH0025pax 1 4.796 5.780 5.398 5.150 5.225 5.626 6.423 JH0026pax 1 4.4916.940 5.363 5.800 6.725 7.646 7.493 JH0027pax 1 3.111 4.720 6.498 4.2805.225 4.486 5.288 JH0028pax 1 4.416 5.680 3.858 5.395 5.820 7.196 6.378JH0029pax 1 4.176 5.950 5.063 5.055 5.850 4.971 6.963 JH0031pax 1 5.3717.385 5.613 6.040 6.595 7.096 7.213 JH0032pax 1 7.326 8.430 4.933 6.0756.925 8.231 8.408 JH0033pax 1 5.258 8.160 5.380 5.560 6.455 8.126 6.181JH0034pax 1 4.061 6.560 4.468 5.725 6.135 6.721 6.703 JH0035pax 1 4.0516.625 3.703 4.690 4.400 6.551 5.523 JH0036pax 1 5.348 6.260 4.608 5.4806.030 6.904 6.618 JH0038pax 1 4.538 6.195 4.878 5.025 5.945 7.049 5.673JH0039pax 1 5.458 6.595 4.165 5.525 6.950 8.691 5.141 JH0040pax 1 5.4586.555 4.773 6.140 6.580 6.144 6.658 JH0041pax 1 6.038 7.940 5.033 6.3356.760 7.489 7.178 JH0042pax 1 3.191 4.985 5.398 4.500 5.978 4.401 4.221JH0043pax 1 5.263 6.670 6.073 6.110 5.955 5.779 9.668 JH0046pax 1 5.1615.360 4.448 5.885 5.808 5.951 5.811 JH0047pax 1 4.396 6.385 4.078 5.6256.828 6.501 6.836 JH0051pax 1 4.881 6.370 5.158 5.290 5.130 5.676 5.388JH0052pax 1 5.066 7.240 5.528 5.510 5.625 5.616 5.703 JH0053pax 1 4.6536.375 5.483 5.258 6.578 5.701 6.571 JH0057pax 1 4.201 7.330 4.208 4.7556.140 6.661 5.968 JH0059pax 1 3.698 4.950 4.243 4.478 4.668 4.896 5.166JH0060pax 1 4.733 6.230 5.303 5.833 6.448 6.646 6.861 JH0061pax 1 5.0637.300 4.298 5.063 6.208 8.041 5.466 JH0063pax 1 4.923 6.845 4.748 5.4335.178 6.021 7.756 JH0065pax 1 3.263 5.220 5.660 4.510 5.355 4.816 4.301JH0066pax 1 5.703 7.575 6.638 5.988 5.818 5.851 6.791 JH0068pax 1 5.5366.448 4.895 5.473 5.925 7.239 7.708 JH0069pax 1 3.723 5.435 4.460 4.8004.955 4.436 4.061 JH0071pax 1 3.748 4.580 6.050 4.785 5.850 5.096 6.261JH0072pax 1 5.863 6.185 5.185 6.015 6.035 7.306 6.386 JH0077pax 1 4.4735.810 5.193 5.635 6.020 5.959 7.278 JH0078pax 1 5.591 5.693 3.685 5.8184.875 5.419 5.778 JH0080pax 1 2.903 4.470 5.033 4.158 5.093 3.921 6.031JH0082pax 1 4.611 5.398 4.800 5.108 5.465 5.364 6.683 JH0083pax 1 3.9036.445 5.398 5.333 4.473 6.006 6.606 JH0086pax 1 5.633 5.850 5.063 5.2884.978 5.936 7.021 JH0092pax 1 5.241 8.328 5.350 6.113 6.540 6.349 7.663MIP0004pax 1 3.201 3.098 5.340 3.873 4.510 4.459 2.743 MP0013Apax 17.028 9.300 6.195 7.105 8.385 8.561 7.406 MP0014Bpax 1 6.418 8.420 6.6236.633 8.008 8.746 7.586 MP0018Apax 1 6.003 7.900 7.005 6.740 6.635 7.1466.281 MP0019Bpax 1 6.283 7.090 6.653 6.528 7.058 6.696 8.656 MP0024pax 15.436 7.823 6.375 6.383 7.655 8.939 8.013 NK2001pax 1 5.061 6.843 6.2305.613 4.680 6.349 7.873 NK2002pax 1 5.516 5.903 5.210 5.568 5.585 6.8096.753 NK2003pax 1 4.986 6.388 5.590 5.588 6.345 7.404 5.723 NK2004pax 14.626 6.648 5.435 5.048 4.945 6.319 5.318

Surprisingly, analysis of the data showed that RNA encoded by ANXA3,CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1 is present on average at asignificantly higher level (p-value less than 0.05) in blood of subjectshaving colorectal cancer relative to subjects having no colorectalpathology, and that RNA encoded by IL2RB is present on average at asignificantly lower level (p-value less than 0.05) in blood of subjectshaving colorectal cancer relative to subjects having no colorectalpathology (Table 5). The ranges of fold-change in the levels of RNAencoded by these genes normalized to levels of RNA encoded by ACTB inblood of the training set subjects having colorectal cancer relative tothe training set subjects not having any colorectal pathology are shownin Table 5.

TABLE 5 Sample training set ranges of fold-changes in levels of RNAencoded by ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1normalized to levels of RNA encoded by ACTB in blood of subjects havingcolorectal cancer relative to subjects not having any colorectalpathology. Gene ANXA3 CLEC4D IL2RB LMNB1 PRRG4 TNFAIP6 VNN1 Averagenormalized RNA 5.21 6.58 5.28 5.76 6.41 6.64 6.77 level in subjectshaving colorectal cancer (ΔCt) Average normalized RNA 5.92 7.02 4.956.09 7.00 17.19 7.31 level in subjects not having any colorectalpathology (ΔCt) Average RNA level fold- 1.63 1.36 0.80 1.26 1.51 1.461.45 change p-value for average RNA level 5.0E−07 2.6E−03 1.1E−035.4E−04 2.3E−06 7.0E−04 1.4E−04 fold-change Maximum observed RNA 11.5315.86 0.07 4.66 6.07 10.12 23.63 level directional fold-change

As can be seen in Table 5, a test subject having a blood level of RNAencoded by ANXA3 which is 1.6 to 11.5 fold higher than the average levelof RNA encoded by this gene in blood of subjects not having anycolorectal pathology is more likely to have colorectal cancer than tonot have any colorectal pathology.

As can be seen in Table 5, a test subject having a blood level of RNAencoded by CLEC4D which is 1.4 to 15.9 fold higher than the averagelevel of RNA encoded by this gene in blood of subjects not having anycolorectal pathology is more likely to have colorectal cancer than tonot have any colorectal pathology.

As can be seen in Table 5, a test subject having a blood level of RNAencoded by LMNB1 which is 1.3 to 4.7 fold higher than the average levelof RNA encoded by this gene in blood of subjects not having anycolorectal pathology is more likely to have colorectal cancer than tonot have any colorectal pathology.

As can be seen in Table 5, a test subject having a blood level of RNAencoded by PRRG4 which is 1.5 to 6.1 fold higher than the average levelof RNA encoded by this gene in blood of subjects not having anycolorectal pathology is more likely to have colorectal cancer than tonot have any colorectal pathology.

As can be seen in Table 5, a test subject having a blood level of RNAencoded by TNFAIP6 which is 1.46 to 10.12 fold higher than the averagelevel of RNA encoded by this gene in blood of subjects not having anycolorectal pathology is more likely to have colorectal cancer than tonot have any colorectal pathology.

As can be seen in Table 5, a test subject having a blood level of RNAencoded by VNN1 which is 1.45 to 23.63 fold higher than the averagelevel of RNA encoded by this gene in blood of subjects not having anycolorectal pathology is more likely to have colorectal cancer than tonot have any colorectal pathology.

As can be seen in Table 5, a test subject having a blood level of RNAencoded by IL2RB which is 0.8 to 0.1 fold that of the average level ofRNA encoded by this gene in blood of subjects not having any colorectalpathology is more likely to have colorectal cancer than to not have anycolorectal pathology.

Generation of Logistic Regression Models for Determining the Probabilitythat a Test Subject has Colorectal Cancer Versus not Having anyColorectal Pathology Via Measurement of Levels of RNA Encoded by ANXA3,CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1 Normalized to Levels ofRNA Encoded by ACTB:

Linear regression analysis of levels of RNA encoded by ANXA3, CLEC4D,IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1 surprisingly showed that logisticregression models based on blood expression levels of all 127 possiblecombinations of one or more of these genes determined in the sampletraining set could be generated, for discriminating, with areceiver-operating characteristic (ROC) area under the curve (AUC) of atleast 0.61, between subjects having colorectal cancer and subjects nothaving any colorectal pathology. Examples of these logistic regressionmodels are shown in Table 6. A model based on ANXA3, CLEC4D, IL2RB,LMNB1, PRRG4, TNFAIP6 and VNN1 (Table 6, Model #1) was surprisinglyfound to enable discrimination with a ROC AUC of 0.79 between subjectshaving colorectal cancer and subjects not having any colorectalpathology.

By way of example, Model #1 of Table 6 corresponds to:

P={1+ê−[0.684+(−0.916)(L _(ANXA3))+(0.353)(L _(CLEC4D))+(0.871)(L_(IL2RB))+(0.907)(L _(LMNB1))+(−0.968)(L _(PRRG4))+(0.154)(L_(TNFAIP6))+(−0.355)(L _(VNN1))]}̂−1,

-   -   where P is the probability that a test subject has colorectal        cancer as opposed to not having any colorectal pathology,        L_(ANXA3) is a ratio of a level of RNA encoded by ANXA3 to a        level of RNA encoded by ACTB in blood of the test subject,        L_(CLEC4D) is a ratio of a level of RNA encoded by CLEC4D to a        level of RNA encoded by ACTB in blood of the test subject,        L_(IL2RB) is a ratio of a level of RNA encoded by IL2RB to a        level of RNA encoded by ACTB in blood of the test subject,        L_(LMNB1) is a ratio of a level of RNA encoded by LMNB1 to a        level of RNA encoded by ACTB in blood of the test subject,        L_(PRRG4) is a ratio of a level of RNA encoded by PRRG4 to a        level of RNA encoded by ACTB in blood of the test subject,        L_(TNFAIP6) is a ratio of a level of RNA encoded by TNFAIP6 to a        level of RNA encoded by ACTB in blood of the test subject, and        L_(VNN1) is a ratio of a level of RNA encoded by VNN1 to a level        of RNA encoded by ACTB in blood of the test subject.

Further by way of example, Model #104 of Table 6 corresponds to:

P={1+ê−[4.311+(−0.659)(L _(PRRG4))]}̂−1,

-   -   where P is the probability that a test subject has colorectal        cancer as opposed to not having any colorectal pathology, and        L_(PRRG4) is a ratio of a level of RNA encoded by PRRG4 to a        level of RNA encoded by ACTB in blood of the test subject.

TABLE 6 Logistic regression models based on blood expression levels ofany possible combination of one or more of ANXA3, CLEC4D, IL2RB, LMNB1,PRRG4, TNFAIP6 and VNN1 for determining the probability that a testsubject has colorectal cancer as opposed to not having colorectalcancer. ROC AUC values for the models are shown for the sample trainingset used to generate the models, as well as for an independent blindsample test set used to test the models. The models, listed in order ofdecreasing ROC AUC value for the training set, are based on expressionlevels determined via quantitative reverse transcriptase-PCR analysisusing ACTB as duplex partner for normalization. The form of these modelsis: P = {1 + e{circumflex over ( )}−[K₀ + K₁L₁ + K₂L₂ + K₃L₃ . . . +K_(n)L_(n)]}{circumflex over ( )}−1, where P is the probability that atest subject has colorectal cancer as opposed to not having anycolorectal pathology; K₀ is a constant; K₁ is a coefficient specific toa first gene; L₁ is a ratio of a level of RNA encoded by the first geneto a level of RNA encoded by ACTB in blood of the test subject; K₂ is acoefficient specific to a second gene; L₂ is a ratio of a level of RNAencoded by the second gene to a level of RNA encoded by ACTB in blood ofthe test subject; K₃ is a coefficient specific to a third gene; L₃ is aratio of a level of RNA encoded by the third gene to a level of RNAencoded by ACTB in blood of the test subject; K_(n) is a coefficientspecific to an nth gene; and L_(n) is a ratio of a level of RNA encodedby the nth gene to a level of RNA encoded by ACTB in blood of the testsubject. No regression coefficients are specified for genes which arenot included in the gene combination (indicated by “—”) on which a givenlogistic regression model is based. No. of ROC AUC Logistic genes BlindGene-specific regression coefficient regression in Training TestConstant (K_(n)) model # model Set Set (K₀) ANXA3 CLEC4D IL2RB LMNB1PRRG4 TNFAIP6 VNN1 1 7 0.79 0.79 0.684 −0.916 0.353 0.871 0.907 −0.968  0.154 −0.355 2 6 0.79 0.79 0.743 −0.859 0.402 0.870 0.893 −0.916 —−0.341 3 4 0.78 0.78 1.321 −0.614 0.358 0.898 — −0.749 — — 4 5 0.78 0.790.343 −0.907 0.322 0.829 0.737 −0.925 — — 5 5 0.78 0.78 1.814 −0.5270.424 0.943 — −0.715 — −0.267 6 5 0.78 0.82 0.830 −0.641 — 0.883 0.935−0.824 — −0.270 7 5 0.78 0.79 1.264 −0.658 0.318 0.898 — −0.788   0.120— 8 5 0.78 0.82 0.359 −0.825 — 0.845 0.794 −0.927   0.189 — 9 6 0.780.80 0.282 −0.953 0.280 0.829 0.740 −0.965   0.123 — 10 6 0.78 0.781.772 −0.575 0.380 0.944 — −0.760   0.141 −0.277 11 6 0.78 0.82 0.727−0.768 — 0.883 0.948 −0.918   0.230 −0.304 12 4 0.77 0.82 0.477 −0.716 —0.848 0.803 −0.850 — — 13 3 0.77 0.80 1.969 — — 1.001 — −0.752 — −0.30514 4 0.77 0.81 1.950 −0.280 — 0.966 — −0.610 — −0.189 15 4 0.77 0.781.915 — 0.161 1.009 — −0.840 — −0.374 16 4 0.77 0.80 1.686 — — 0.9950.238 −0.855 — −0.363 17 5 0.77 0.79 1.748 — 0.130 1.005 0.147 −0.887 —−0.397 18 4 0.77 0.82 1.436 −0.487 — 0.925 — −0.731   0.195 — 19 4 0.770.80 1.934 — — 1.008 — −0.808   0.084 −0.334 20 5 0.77 0.81 1.861 −0.398— 0.965 — −0.697   0.223 −0.218 21 5 0.77 0.79 1.907 — 0.147 1.011 —−0.852   0.029 −0.378 22 5 0.77 0.79 1.697 — — 1.001 0.207 −0.880  0.058 −0.376 23 6 0.77 0.79 1.748 — 0.121 1.006 0.142 −0.894   0.021−0.399 24 2 0.76 0.79 1.225 — — 0.957 — −0.928 — — 25 3 0.76 0.81 1.570−0.371 — 0.930 — −0.649 — — 26 3 0.76 0.79 1.233 — −0.006   0.957 —−0.924 — — 27 3 0.76 0.79 1.328 — — 0.960 −0.058   −0.894 — — 28 4 0.760.79 1.332 — 0.015 0.960 −0.072   −0.898 — — 29 3 0.76 0.79 1.249 — —0.956 — −0.912 −0.019 — 30 4 0.76 0.79 1.244 — 0.006 0.956 — −0.914−0.022 — 31 4 0.76 0.79 1.328 — — 0.959 −0.052   −0.890 −0.009 — 32 50.76 0.79 1.333 — 0.022 0.960 −0.067   −0.893 −0.016 — 33 3 0.75 0.800.744 −0.544 — 0.820 — — — −0.282 34 4 0.75 0.78 0.536 −0.717 0.2520.784 — — — −0.333 35 4 0.75 0.80 0.324 −0.670 — 0.780 0.260 — — −0.31336 5 0.75 0.78 0.241 −0.798 0.236 0.757 0.189 — — −0.353 37 5 0.75 0.752.890 −1.124 0.436 — 1.167 −0.723 — −0.254 38 4 0.75 0.80 0.701 −0.567 —0.818 — —   0.033 −0.288 39 5 0.75 0.78 0.571 −0.702 0.266 0.785 — —−0.036 −0.330 40 5 0.75 0.80 0.308 −0.680 — 0.779 0.254 —   0.019 −0.31641 6 0.75 0.78 0.271 −0.784 0.253 0.757 0.198 — −0.043 −0.349 42 6 0.750.76 2.851 −1.176 0.395 — 1.177 −0.767   0.134 −0.266 43 2 0.74 0.800.043 −0.713 — 0.751 — — — — 44 3 0.74 0.79 −0.169 −0.843 0.160 0.721 —— — — 45 3 0.74 0.80 −0.101 −0.755 — 0.738 0.076 — — — 46 4 0.74 0.79−0.190 −0.848 0.159 0.719 0.011 — — — 47 4 0.74 0.75 2.528 −1.146 0.378— 1.036 −0.741 — — 48 4 0.74 0.77 1.234 — −0.009   0.883 −0.489   — —−0.407 49 3 0.74 0.80 0.070 −0.701 — 0.753 — — −0.015 — 50 4 0.74 0.78−0.082 −0.811 0.189 0.723 — — −0.068 — 51 4 0.74 0.80 −0.079 −0.742 —0.738 0.084 — −0.021 — 52 5 0.74 0.78 −0.130 −0.824 0.187 0.719 0.028 —−0.069 — 53 5 0.74 0.76 2.481 −1.189 0.342 — 1.038 −0.778   0.110 — 54 40.74 0.77 1.245 — — 0.878 −0.387   — −0.120 −0.382 55 5 0.74 0.77 1.272— 0.059 0.877 −0.424   — −0.139 −0.392 56 3 0.73 0.77 1.238 — — 0.883−0.498   — — −0.409 57 4 0.73 0.80 3.012 −0.891 — — 1.247 −0.638 —−0.182 58 5 0.73 0.80 2.927 −1.011 — — 1.251 −0.721   0.217 −0.212 59 30.72 0.73 4.212 −0.733 0.451 — — −0.493 — — 60 3 0.72 0.80 2.728 −0.930— — 1.141 −0.660 — — 61 3 0.72 0.77 0.438 — −0.175   0.826 — — — −0.50562 4 0.72 0.73 4.547 −0.690 0.489 — — −0.466 — −0.144 63 3 0.72 0.760.855 — — 0.835 −0.659   — −0.188 — 64 4 0.72 0.77 0.847 — −0.031  0.836 −0.636   — −0.177 — 65 4 0.72 0.74 4.171 −0.772 0.418 — — −0.527  0.104 — 66 4 0.72 0.80 2.614 −1.040 — — 1.128 −0.735   0.188 — 67 30.72 0.76 0.608 — — 0.823 — — −0.217 −0.482 68 4 0.72 0.77 0.637 —−0.049   0.828 — — −0.194 −0.466 69 5 0.72 0.74 4.526 −0.731 0.455 — —−0.504   0.117 −0.154 70 2 0.71 0.76 0.786 — — 0.840 −0.871   — — — 71 30.71 0.78 0.770 — −0.122   0.844 −0.733   — — — 72 2 0.71 0.74 0.162 — —0.799 — — — −0.616 73 4 0.71 0.74 2.134 −1.028 0.310 — 0.571 — — −0.28674 5 0.71 0.74 2.164 −1.014 0.327 — 0.580 — −0.044 −0.282 75 3 0.70 0.751.725 −1.054 0.242 — 0.404 — — — 76 2 0.70 0.80 4.710 −0.417 — — —−0.372 — — 77 3 0.70 0.73 3.283 −0.791 0.371 — — — — −0.214 78 3 0.700.79 2.309 −0.868 — — 0.682 — — −0.231 79 3 0.70 0.80 4.842 −0.393 — — —−0.360 — −0.050 80 4 0.70 0.75 1.785 −1.031 0.269 — 0.421 — −0.069 — 813 0.70 0.80 4.558 −0.539 — — — −0.455   0.200 — 82 4 0.70 0.73 3.305−0.783 0.378 — — — −0.020 −0.211 83 4 0.70 0.79 2.279 −0.886 — — 0.671 —  0.034 −0.237 84 4 0.70 0.81 4.759 −0.506 — — — −0.440   0.211 −0.07985 1 0.69 0.79 3.324 −0.616 — — — — — — 86 2 0.69 0.74 2.681 −0.8680.301 — — — — — 87 2 0.69 0.79 1.931 −0.919 — — 0.520 — — — 88 2 0.690.79 3.768 −0.536 — — — — — −0.126 89 2 0.69 0.79 3.214 −0.654 — — — —  0.047 — 90 3 0.69 0.73 2.748 −0.847 0.321 — — — −0.046 — 91 2 0.690.73 −0.674 — — 0.701 — — −0.435 — 92 3 0.69 0.76 −0.326 — −0.240  0.745 — — −0.281 — 93 3 0.69 0.79 1.931 −0.919 — — 0.520 —   0.000 — 943 0.69 0.80 3.648 −0.587 — — — —   0.075 −0.143 95 2 0.68 0.75 −0.764 —−0.455   0.736 — — — — 96 2 0.68 0.78 4.977 — — — — −0.524 — −0.223 97 30.68 0.76 4.939 — 0.113 — — −0.579 — −0.275 98 3 0.68 0.77 4.670 — — —0.238 −0.620 — −0.289 99 4 0.68 0.76 4.716 — 0.067 — 0.185 −0.631 —−0.304 100 3 0.68 0.78 4.975 — — — — −0.531   0.012 −0.228 101 4 0.680.76 4.941 — 0.134 — — −0.564 −0.042 −0.268 102 4 0.68 0.77 4.655 — — —0.255 −0.610 −0.028 −0.283 103 5 0.68 0.76 4.702 — 0.091 — 0.200 −0.615−0.055 −0.299 104 1 0.67 0.76 4.311 — — — — −0.659 — — 105 2 0.67 0.774.327 — −0.010   — — −0.652 — — 106 2 0.67 0.76 4.314 — — — −0.001  −0.658 — — 107 3 0.67 0.77 4.309 — −0.014   — 0.012 −0.656 — — 108 20.67 0.77 4.391 — — — — −0.607 −0.062 — 109 3 0.67 0.76 4.356 — 0.035 —— −0.619 −0.079 — 110 3 0.67 0.76 4.292 — — — 0.056 −0.629 −0.074 — 1114 0.67 0.76 4.299 — 0.024 — 0.038 −0.630 −0.082 — 112 3 0.65 0.74 3.919— — — −0.142   — −0.152 −0.303 113 2 0.64 0.74 3.402 — −0.105   — — — —−0.397 114 2 0.64 0.75 3.927 — — — −0.277   — — −0.340 115 3 0.64 0.753.922 — −0.010   — −0.268   — — −0.337 116 2 0.64 0.73 3.528 — — —−0.373   — −0.206 — 117 3 0.64 0.73 3.528 — — — −0.373   — −0.206 — 1182 0.64 0.73 3.628 — — — — — −0.189 −0.345 119 3 0.64 0.73 3.610 — 0.020— — — −0.199 −0.352 120 4 0.64 0.73 3.951 — 0.071 — −0.188   — −0.174−0.316 121 1 0.63 0.75 3.466 — — — −0.603   — — — 122 2 0.63 0.78 3.457— −0.104   — −0.482   — — — 123 1 0.63 0.71 3.191 — — — — — — −0.468 1242 0.63 0.74 2.657 — −0.133   — — — −0.269 — 125 1 0.62 0.69 2.363 — — —— — −0.357 — 126 1 0.61 0.73 2.197 — −0.338   — — — — — 127 1 0.61 0.64−2.973 — — 0.561 — — — —

Blind Sample Test Set:

Quantitative RT-PCR analysis of gene expression in an independent blindtest set of blood samples from 76 subjects having colorectal cancer and77 subjects not having any colorectal pathology was performed asdescribed above for the training set. The normalized RNA levels measuredare shown in Table 7.

TABLE 7 Sample test set levels of RNA encoded by ANXA3, CLEC4D, IL2RB,LMNB1, PRRG4, TNFAIP6 and VNN1 in blood of subjects having colorectalcancer (Group 1) and subjects not having any colorectal pathology (Group0), normalized to levels of RNA encoded by ACTB. Levels shown correspondto ΔCt. Gene Sample ID Group ANXA3 CLEC4D IL2RB LMNB1 PRRG4 TNFAIP6 VNN1CD0214pax 0 5.56 6.17 4.50 6.44 7.07 5.78 6.69 CD0242pax 0 5.87 7.324.54 5.43 5.19 5.87 7.38 RC2897pax 0 6.67 8.37 5.49 5.95 7.18 7.80 7.96CD0670pax 0 7.79 5.80 3.25 6.35 7.10 8.40 7.80 CD1401pax 0 5.84 6.395.42 5.85 7.30 6.55 7.12 PB2924pax 0 5.56 7.17 4.12 5.97 6.51 5.90 7.12CD0482pax 0 7.65 8.34 3.43 6.91 7.02 8.94 8.48 PB1275pax 0 5.26 6.135.18 5.54 7.09 5.89 8.78 CD0148pax 0 5.90 7.83 3.98 5.95 7.60 7.35 7.20CD0122pax 0 6.55 8.40 5.02 6.38 7.07 7.96 8.83 PB2272pax 0 7.30 8.303.95 6.57 7.42 9.42 7.49 CD1708pax 0 6.28 9.11 4.07 5.74 6.01 7.48 7.13CD0354pax 0 6.55 8.20 4.93 6.30 8.06 8.28 7.81 PB2634pax 0 5.76 7.745.28 6.80 7.20 9.18 7.39 CD0204pax 0 5.90 6.53 4.83 5.56 6.25 7.29 6.87PB1336pax 0 7.60 9.00 6.87 6.99 7.13 8.92 9.71 RC2699pax 0 7.72 9.426.03 7.12 7.99 8.67 8.57 CD1278pax 0 5.95 7.57 4.20 5.72 6.31 6.83 7.36PB2062pax 0 7.31 7.76 4.31 6.50 7.29 8.38 7.78 PB2464pax 0 5.95 8.334.73 5.52 5.52 6.39 8.43 CD0053pax 0 6.17 7.90 4.09 6.56 7.18 8.25 7.44CD0192pax 0 5.49 7.60 4.19 5.66 7.38 5.48 6.03 CD0244pax 0 6.32 7.315.26 6.21 7.56 7.63 6.89 CD0833pax 0 5.52 8.65 4.72 5.55 6.33 6.60 5.47CD1719pax 0 6.19 7.94 5.40 5.93 6.39 6.50 7.51 CD0036pax 0 5.20 5.954.60 4.95 4.51 5.99 5.82 PB2015pax 0 6.62 7.13 3.85 5.99 7.62 8.53 7.13PB0662pax 0 6.09 7.17 5.33 6.49 7.40 7.43 6.60 PB2024pax 0 6.16 6.104.49 5.94 6.86 8.23 7.44 RC2565pax 0 6.63 8.81 5.94 6.33 8.38 7.11 8.75CD1561pax 0 7.29 6.78 4.73 6.48 7.49 8.18 8.21 CD1728pax 0 7.13 6.894.30 6.52 7.76 7.08 8.63 CD0238pax 0 5.47 7.06 4.85 6.10 7.13 7.38 7.14PB2342pax 0 7.65 4.19 6.21 7.29 8.02 8.00 CD0800pax 0 7.42 8.19 3.996.61 7.78 8.79 8.44 CD0437pax 0 4.74 5.92 5.05 4.90 5.58 6.27 6.17RC3214pax 0 6.35 8.13 4.96 6.22 7.02 8.19 7.01 CD1487pax 0 5.24 7.834.51 5.99 7.26 7.36 6.48 PB1763pax 0 7.83 9.12 5.21 7.40 8.78 9.20 8.37CD0580pax 0 5.14 5.72 3.17 4.54 4.78 6.28 5.28 CD0840pax 0 4.94 6.634.96 5.38 5.60 6.63 6.10 PB2757pax 0 5.32 7.54 4.98 5.51 6.48 6.77 8.12PB2184pax 0 5.11 7.34 5.73 5.68 6.63 6.30 8.26 PB2179pax 0 5.57 6.844.80 5.59 5.75 5.74 6.56 PB1324pax 0 4.77 9.26 4.24 5.57 6.78 7.16 7.37CD0237pax 0 6.46 8.06 5.42 5.89 6.89 7.51 7.26 CD1329pax 0 6.03 6.724.93 5.93 6.06 7.37 7.38 PB2005pax 0 7.51 7.37 4.66 6.41 7.63 9.46 8.15PB3227pax 0 5.08 6.46 4.44 5.74 6.46 7.62 7.51 PB3163pax 0 4.51 6.234.17 5.32 6.10 7.12 6.54 PB3481pax 0 5.66 7.99 5.16 5.95 8.33 6.48 8.26CD1320pax 0 4.16 5.93 4.79 4.60 5.55 5.10 6.16 RC3191pax 0 7.15 7.644.92 6.94 7.20 8.20 8.30 CD0583pax 0 4.29 7.00 4.44 4.16 3.96 8.82 3.97PB3032pax 0 5.12 6.51 5.09 5.96 6.08 6.91 7.87 CD0367pax 0 5.75 6.684.34 5.49 6.25 7.12 7.06 PB2889pax 0 6.03 8.25 4.75 6.39 7.27 7.04 7.68PB3524pax 0 6.66 6.73 5.40 6.83 7.72 6.07 7.38 RC2986pax 0 6.90 7.965.36 6.14 7.22 8.87 7.75 CD1428pax 0 6.64 9.25 3.67 5.82 7.04 7.12 6.09RC2236pax 0 6.82 6.87 5.24 6.21 7.41 7.27 8.02 PB1918pax 0 8.72 7.415.20 7.28 8.29 10.44 9.02 CD0277pax 0 6.42 8.60 5.37 6.63 7.61 8.57 7.04CD0667pax 0 5.15 6.09 5.57 5.26 6.33 6.27 6.41 CD1741pax 0 5.46 6.185.57 5.76 6.50 7.24 7.18 PB1973pax 0 8.07 9.15 4.68 7.12 8.89 9.13 10.01PB1222pax 0 5.80 7.45 5.57 5.95 5.81 6.15 7.61 RC2683pax 0 8.25 9.295.83 6.98 8.07 9.46 9.66 PB3200pax 0 5.00 6.73 4.23 5.42 6.59 6.31 7.35PB2130pax 0 6.31 7.61 4.91 5.50 5.79 6.48 6.81 PB3097pax 0 6.33 3.945.77 6.08 6.72 7.78 7.51 CD0571pax 0 5.28 6.04 3.76 5.54 6.60 6.99 5.82CD0676pax 0 5.50 6.68 5.78 5.79 5.91 6.91 7.18 PB1514pax 0 7.57 5.965.35 7.38 8.09 7.63 8.37 CD0547pax 0 4.62 7.13 6.05 5.85 7.44 6.70 6.88CD1068pax 0 5.79 6.49 4.84 6.23 7.14 8.55 6.32 CD0715pax 0 2.97 4.034.74 4.51 4.94 5.38 6.46 JH0130pax 1 5.32 5.90 6.01 5.47 5.77 5.93 6.90MH0079pax 1 6.41 6.17 5.06 6.19 6.42 6.61 7.57 MH0082pax 1 7.06 4.935.26 6.82 6.74 8.27 7.32 AN0013pax 1 4.94 7.96 3.89 5.60 5.49 7.86 7.95NK2005pax 1 4.20 5.83 5.59 5.08 5.65 4.02 6.98 CD1111pax 1 6.62 6.644.17 6.17 7.32 7.62 7.72 JH0105pax 1 5.31 7.29 4.84 5.39 5.34 7.13 8.28MIP1007pax 1 3.42 7.30 4.89 4.27 6.00 5.13 3.62 DC0011pax 1 4.33 5.634.17 5.45 5.74 6.41 7.06 MH0073pax 1 4.68 6.28 5.84 4.57 5.52 6.41 5.86DC0003pax 1 6.24 7.65 5.02 6.05 6.64 7.05 8.46 DC1002pax 1 4.62 4.355.41 5.26 6.40 7.24 6.90 JH0096pax 1 3.45 5.49 5.18 4.79 5.74 3.34 7.61KW0002pax 1 4.26 6.89 4.62 4.71 4.76 5.78 6.53 JH0120pax 1 5.89 4.915.53 6.38 5.56 8.45 6.44 MIP1008pax 1 4.84 7.74 5.47 5.41 5.63 5.36 6.93MIP0002pax 1 3.76 6.96 5.63 4.85 6.55 5.74 6.87 MIP1011pax 1 4.70 6.285.12 4.63 4.60 5.68 4.55 MH0074pax 1 5.11 5.73 5.75 5.68 6.74 7.59 6.00DC0008pax 1 4.97 6.33 4.16 5.44 4.95 6.54 5.87 AN4014pax 1 3.93 5.764.25 4.58 5.76 5.20 7.74 DC0012pax 1 4.25 6.07 5.15 5.08 7.00 4.86 6.37MIP2002pax 1 5.54 5.71 4.26 5.54 5.77 6.57 6.33 NK1005pax 1 5.76 8.795.22 7.02 7.38 8.60 7.55 MIP0007pax 1 2.91 3.55 6.69 3.21 4.38 3.48 4.85JH0118pax 1 5.72 7.53 4.77 5.56 5.85 6.56 6.04 JH0089pax 1 5.11 7.304.99 5.68 5.58 6.54 7.38 MH0057pax 1 5.80 6.60 6.01 6.35 6.36 7.44 6.44DC0005pax 1 5.98 7.66 4.72 6.38 6.39 7.47 7.19 MH0067pax 1 4.75 6.066.11 4.96 5.30 6.78 5.54 JH0085pax 1 5.19 6.48 5.92 5.99 5.76 6.65 7.12JH0127pax 1 5.48 6.43 5.18 6.13 7.59 6.63 7.04 MIP1013pax 1 4.86 5.894.56 5.29 5.21 5.89 6.51 JH0126pax 1 4.21 7.08 5.35 5.68 5.40 5.50 7.07AN4013pax 1 5.15 5.66 4.15 5.19 5.38 5.94 6.91 MH0053pax 1 7.07 8.104.16 6.42 5.83 8.33 7.84 JH0115pax 1 3.82 7.54 5.74 4.75 6.03 6.27 7.36CC0004pax 1 3.83 5.81 4.59 4.67 5.46 5.50 5.84 JH0091pax 1 5.26 5.924.74 5.31 7.34 6.70 6.91 NK1004pax 1 5.76 7.32 6.16 5.78 6.58 7.99 7.77NK1008pax 1 4.70 7.52 5.56 5.57 6.80 6.58 7.52 MIP2006pax 1 4.25 6.384.28 4.74 5.69 5.36 5.38 AN0020pax 1 4.12 6.61 5.57 4.81 4.61 4.45 6.07JH0117pax 1 3.36 8.59 4.90 4.29 4.99 5.12 5.76 JH0100pax 1 5.54 6.694.86 5.66 6.57 7.23 7.26 MH0066pax 1 5.12 5.25 6.28 5.59 6.61 6.27 6.13NK2009pax 1 5.30 5.93 4.95 5.76 5.27 7.71 5.77 NK2008pax 1 4.14 5.575.88 4.93 5.45 6.59 5.66 PB3067-2pax 1 5.84 8.25 4.70 6.20 6.51 7.786.81 NK1003pax 1 4.41 6.99 5.48 5.30 6.08 6.48 7.13 MIP1009pax 1 3.228.02 6.45 4.25 5.14 5.47 5.21 DC2006pax 1 6.06 5.91 4.81 5.88 6.05 7.247.34 JH0131pax 1 5.19 6.71 4.98 5.21 5.15 4.75 6.43 DC0015pax 1 4.956.98 5.01 5.46 6.53 6.02 6.17 AN0001pax 1 4.36 6.54 5.97 5.60 5.83 6.727.13 JH0111pax 1 5.04 6.83 4.22 5.09 6.96 7.14 5.78 MIP0005pax 1 3.745.56 5.98 4.46 5.80 5.74 5.33 MH0065pax 1 4.50 5.37 5.63 5.51 6.30 6.095.82 JH0136pax 1 3.39 4.63 6.12 4.86 5.19 7.76 4.59 CD1351pax 1 6.867.94 5.55 6.06 6.52 8.46 7.62 MH0075pax 1 6.05 7.17 5.57 5.77 6.75 7.327.27 MH0078pax 1 4.60 6.70 5.49 5.26 4.21 5.25 7.54 MH0068pax 1 7.585.49 4.48 6.43 7.14 8.00 6.36 MIP2003pax 1 4.24 5.60 4.30 4.93 6.05 6.855.97 NK2015pax 1 5.42 5.52 5.54 6.08 6.40 8.40 8.09 MH0070pax 1 5.407.11 4.66 5.74 6.55 7.09 7.08 JH0093pax 1 5.22 7.62 4.66 5.29 5.39 7.007.15 JH0135pax 1 4.40 4.64 5.52 4.62 5.38 4.31 4.62 CD1571pax 1 5.456.48 4.41 5.65 7.26 7.88 7.17 MH0061pax 1 4.99 5.15 5.47 5.72 6.73 7.657.76 NK2007pax 1 5.98 7.25 5.17 5.76 7.02 7.32 6.38 JH0132pax 1 5.297.18 5.29 5.76 6.96 6.66 6.83 MH0062pax 1 4.40 6.01 4.86 5.42 5.50 6.266.45 JH0114pax 1 4.78 6.93 7.12 5.58 7.50 5.62 5.26 CD1260pax 1 5.285.92 5.24 5.73 5.86 7.18 7.37 JH0022pax 1 5.00 5.94 4.73 5.49 6.17 5.666.80

Analysis of the test set results confirmed the surprising finding basedon the training set that ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 andVNN1 each express RNA on average at a significantly higher level(p-value less than 0.05) in blood of subjects having colorectal cancerrelative to subjects having no colorectal pathology, and that IL2RBexpresses RNA on average at a significantly lower level (p-value lessthan 0.05) in blood of subjects having colorectal cancer relative tosubjects having no colorectal pathology (Table 8). The ranges offold-change in the levels of RNA encoded by ANXA3, CLEC4D, IL2RB, LMNB1,PRRG4, TNFAIP6 and VNN1 normalized to levels of RNA encoded by ACTB inblood of the test set subjects having colorectal cancer relative to thetest set subjects not having any colorectal pathology are also shown inTable 8.

TABLE 8 Sample test set ranges of fold-change in levels of RNA encodedby ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6, VNN1 normalized tolevels of RNA encoded by ACTB in blood of subjects having colorectalcancer relative to subjects not having any colorectal pathology. GeneANXA3 CLEC4D IL2RB LMNB1 PRRG4 TNFAIP6 VNN1 Average normalized 4.98 6.455.18 5.42 6.01 6.51 6.63 RNA level in subjects having colorectal cancer(ΔCt) Average normalized 6.10 7.32 4.83 6.01 6.86 7.41 7.40 RNA level insubjects not having any colorectal pathology (ΔCt) Average RNA level2.17 1.82 0.78 1.50 1.80 1.87 1.70 fold-change p-value for average1.7E−10 1.9E−06 1.4E−03 1.3E−07 1.6E−08 3.8E−06 4.4E−06 RNA level fold-change Maximum observed 9.13 13.66 0.20 6.98 6.26 16.78 13.78 RNA leveldirectional fold-change

As can be seen in Table 8, a test subject having a blood level of RNAencoded by ANXA3 which is 2.2 to 9.1 fold higher than the average levelof RNA encoded by this gene in blood of subjects not having anycolorectal pathology is more likely to have colorectal cancer than tonot have any colorectal pathology.

As can be seen in Table 8, a test subject having a blood level of RNAencoded by CLEC4D which is 1.8 to 13.7 fold higher than the averagelevel of RNA encoded by this gene in blood of subjects not having anycolorectal pathology is more likely to have colorectal cancer than tonot have any colorectal pathology.

As can be seen in Table 8, a test subject having a blood level of RNAencoded by LMNB1 which is 1.5 to 7.0 fold higher than the average levelof RNA encoded by this gene in blood of subjects not having anycolorectal pathology is more likely to have colorectal cancer than tonot have any colorectal pathology.

As can be seen in Table 8, a test subject having a blood level of RNAencoded by PRRG4 which is 1.8 to 6.3 fold higher than the average levelof RNA encoded by this gene in blood of subjects not having anycolorectal pathology is more likely to have colorectal cancer than tonot have any colorectal pathology.

As can be seen in Table 8, a test subject having a blood level of RNAencoded by TNFAIP6 which is 1.9 to 16.8 fold higher than the averagelevel of RNA encoded by this gene in blood of subjects not having anycolorectal pathology is more likely to have colorectal cancer than tonot have any colorectal pathology.

As can be seen in Table 8, a test subject having a blood level of RNAencoded by VNN1 which is 1.7 to 13.8 fold higher than the average levelof RNA encoded by this gene in blood of subjects not having anycolorectal pathology is more likely to have colorectal cancer than tonot have any colorectal pathology.

As can be seen in Table 8, a test subject having a blood level of RNAencoded by IL2RB which is 0.8 to 0.2 fold that of the average level ofRNA encoded by this gene in blood of subjects not having any colorectalpathology is more likely to have colorectal cancer than to not have anycolorectal pathology.

Furthermore, the test set results confirmed the surprising finding basedon the training set that logistic regression models based on bloodexpression levels for any of the 127 possible combinations of one ormore of ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1, each ofwhich normalized against expression levels of ACTB, can be used todiscriminate, with a ROC AUC of at least 0.64 (Table 6), betweensubjects having colorectal cancer and subjects not having any colorectalpathology. As such, the novel logistic regression models listed in Table6 can be used to determine the probability that a test subject hascolorectal cancer as opposed to not having any colorectal pathology,based on blood levels of expression of ANXA3, CLEC4D, IL2RB, LMNB1,PRRG4, TNFAIP6 and/or VNN1.

Example 3 Measurement of Blood Levels of RNA Encoded by any Combinationof ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and/or VNN1 Relative to theLevel of RNA Encoded by IL2RB can be Used to Determine the Probabilitythat a Test Subject has Colorectal Cancer as Opposed to not Having anyColorectal Pathology

Materials and Methods:

Refer to “General materials and methods”, above.

Experimental Results:

Sample Training Set:

Discovery of Significantly Different Levels of RNA Encoded by ANXA3,CLEC4D, LMNB1, PRRG4, VNN1, TNFAIP6 Normalized to IL2RB in Blood ofSubjects Having Colorectal Cancer Relative to Subjects not Having anyColorectal Pathology:

Quantitative reverse transcriptase-PCR analysis of gene expression in atraining set of blood samples from 116 subjects having colorectal cancerand 127 subjects not having any colorectal pathology, using IL2RB asduplex partner for normalization of gene expression levels wasperformed. The normalized RNA levels measured are shown in Table 9

TABLE 9 Sample training set levels of RNA encoded by ANXA3, CLEC4D,IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1 in blood of subjects havingcolorectal cancer (Group 1) and subjects not having any colorectalpathology (Group 0), normalized to levels of RNA encoded by IL2RB.Levels shown correspond to ΔCt. Gene Sample ID Group ANXA3 CLEC4D LMNB1PRRG4 TNFAIP6 VNN1 CD0011pax 0 0.8303 1.3467 1.2008 0.8909 2.1665 2.6036CD0012pax 0 1.2503 1.4917 0.8258 0.9309 1.9115 2.9086 CD0030pax 0 1.28781.2957 0.7182 1.1632 0.7535 1.7757 CD0063pax 0 2.4078 3.0807 1.63321.8532 3.4685 3.0157 CD0077pax 0 −0.0047 1.2417 0.7058 0.7009 0.84651.7436 CD0078pax 0 1.6928 2.4057 0.9432 0.9332 2.6735 1.4807 CD0085pax 00.4428 1.5007 0.4032 0.8232 2.4335 0.4457 CD0117pax 0 0.6028 2.20570.7632 2.0182 2.2585 0.6557 CD0146pax 0 0.0353 0.7117 0.6708 0.16591.3215 0.7236 CD0167pax 0 −1.3147 −0.1483 0.0358 0.2609 1.3315 0.0786CD0249pax 0 −0.7797 −0.6233 −0.8142 0.0609 −0.0085 0.2486 CD0279pax 00.8278 1.9907 0.7632 0.7382 1.6185 1.4957 CD0286pax 0 0.0753 1.52170.7058 0.6659 0.7215 0.7286 CD0297pax 0 −0.1797 0.2867 0.1958 −0.32910.0665 0.4836 CD0323pax 0 1.7303 1.6817 0.5408 1.2359 2.2865 3.0636CD0445pax 0 0.9878 1.2107 0.6682 0.6932 1.8085 1.6907 CD0463pax 0−0.3647 1.2267 0.3058 0.5559 1.2365 1.0136 CD0491pax 0 −0.3972 0.35070.4482 0.7032 1.1785 0.2907 CD0496pax 0 0.8753 2.4167 0.6958 1.45091.0515 0.6436 CD0501pax 0 0.8753 1.7517 0.9558 0.9109 2.7615 1.0986CD0504pax 0 −0.4947 0.2367 0.2358 1.0209 0.3715 0.7936 CD0573pax 01.9978 1.3107 0.9582 0.9982 2.4435 1.9357 CD0578pax 0 1.6103 1.73170.6408 0.6259 1.6865 1.7136 CD0639pax 0 0.1028 0.6657 0.6232 −0.26180.9985 1.5857 CD0645pax 0 −0.8347 −0.1883 −0.4342 −0.4441 0.4965 0.0986CD0679pax 0 0.4703 1.7767 0.6658 0.5659 1.9465 2.2636 CD0685pax 0 0.87532.3317 0.8008 1.3759 2.1915 1.4836 CD0716pax 0 1.9978 2.4057 0.91820.4432 1.5835 2.0457 CD0749pax 0 0.3303 1.3717 0.4058 0.5509 2.04651.0336 CD0760pax 0 −2.5747 −1.3583 −1.9442 −1.1491 −1.2335 −0.8714CD0811pax 0 2.2353 2.8417 1.1858 1.3609 1.3865 1.3286 CD0846pax 0−0.6522 0.3407 −0.6418 −0.3468 −0.6315 1.5657 CD0848pax 0 0.7278 1.20570.9832 1.0232 1.5785 1.8307 CD0924pax 0 0.1428 0.5357 −0.0768 −0.44180.2935 −0.0793 CD1066pax 0 0.1753 0.6667 0.2658 0.2059 1.9715 0.5786CD1073pax 0 0.1053 −0.0633 −0.0092 −0.1341 0.8015 1.0886 CD1075pax 0−0.3322 0.4757 −0.0118 1.1582 1.2385 0.1957 CD1089pax 0 −1.1072 0.1657−0.7818 −0.5318 0.1935 −0.1193 CD1116pax 0 −0.2072 −0.0543 −0.28180.2982 1.4885 0.4107 CD1120pax 0 −0.8872 0.0357 −0.5868 0.2032 1.1885−0.2443 CD1198pax 0 0.5253 0.5317 0.7908 0.7509 0.7915 0.9786 PB1179pax0 1.0003 1.0617 0.8808 0.9909 2.4315 1.2286 PB1277pax 0 0.8803 1.48170.4258 0.9859 1.5765 0.8286 PB1301pax 0 −1.7247 −0.9183 −0.9092 −0.5091−0.6985 −1.2864 PB1315pax 0 −1.5572 0.2257 −0.6218 −0.3118 −0.20150.9007 PB1345pax 0 0.1953 0.6617 0.7508 1.0459 0.3615 2.0236 PB1518pax 01.2653 1.6967 0.2058 0.8359 1.3565 1.3036 PB1520pax 0 1.0953 2.02670.9708 0.0109 1.6565 2.2236 PB1574pax 0 1.0753 1.5467 0.8008 0.61091.0465 1.1836 PB1783pax 0 1.5978 1.7007 1.1982 1.2632 2.1135 1.5907PB1799pax 0 0.6978 1.0157 0.5282 1.0632 2.0785 0.5957 PB1811pax 0 0.86281.4057 0.8232 1.2632 1.3685 0.9507 PB1830pax 0 0.1428 0.6807 0.25320.1182 0.8985 2.3407 PB1833pax 0 0.2028 1.1407 0.4782 0.5532 0.93851.5457 PB1843pax 0 0.5553 0.4717 0.0708 0.5909 −0.5285 1.1486 PB1851pax0 0.4428 0.1007 0.2632 0.3232 2.7035 1.6007 PB1919pax 0 1.4703 2.10670.8108 1.7959 2.1365 2.5086 PB1922pax 0 0.3103 1.1867 0.4258 1.37592.1215 0.9986 PB1924pax 0 0.2428 0.5357 0.1632 0.3632 1.1535 1.2357PB1937pax 0 1.3128 2.1057 1.0982 2.9132 2.4835 2.2007 PB1964pax 0 0.48282.6207 0.7382 1.5332 2.4035 2.7507 PB2027pax 0 −0.1422 0.3857 0.15820.3182 1.1835 1.5807 PB2029pax 0 0.1953 0.5917 0.0708 −0.0391 1.86150.7336 PB2073pax 0 0.4478 0.9057 0.4382 1.6632 1.5085 0.9257 PB2086pax 00.1153 0.1817 0.3108 0.8409 0.3215 0.5386 PB2099pax 0 −0.4622 −0.5993−0.1568 0.0732 −0.1265 0.1057 PB2100pax 0 1.1628 1.1057 0.6532 1.54822.1385 1.1507 PB2132pax 0 0.5503 1.1517 0.3558 0.6109 2.1315 1.4536PB2168pax 0 1.3278 1.5357 0.8482 1.1682 2.3435 1.3307 PB2192pax 0 0.31530.8967 0.3308 −0.0291 1.2515 1.9286 PB2196pax 0 0.8328 1.6107 0.79321.4432 2.0535 1.2107 PB2200pax 0 1.1028 1.4807 0.3732 0.4432 0.96350.5757 PB2213pax 0 2.1753 2.2717 1.1658 0.9009 3.1465 1.9736 PB2224pax 0−0.2772 0.6207 0.0482 0.3582 2.1235 1.2607 PB2228pax 0 1.7703 2.10171.3658 1.6859 3.6965 2.7186 PB2229pax 0 −0.2047 −0.3183 −0.0542 −0.92410.5615 −0.1764 PB2277pax 0 1.3578 0.8507 0.6332 0.3582 1.0785 1.7457PB2297pax 0 0.4178 1.1507 0.1282 0.0332 0.9235 −0.3343 PB2312pax 01.3628 2.0057 1.4132 0.9632 2.0885 2.0357 PB2398pax 0 −0.5822 0.1007−0.2868 −0.7518 −0.1865 1.3307 PB2409pax 0 0.1153 0.6817 0.3008 0.33591.0115 1.5036 PB2414pax 0 2.7128 2.3707 1.2132 2.1832 3.6985 1.9707PB2467pax 0 0.7478 1.1457 0.5482 0.6382 2.2385 1.3607 PB2473pax 0 0.38280.8857 0.4432 1.0932 0.9185 1.8407 PB2512pax 0 0.7003 1.7267 0.84580.4709 1.8115 2.0136 PB2568pax 0 −0.4772 0.0157 −0.3718 0.4132 1.24351.0857 PB2571pax 0 0.3003 1.0017 0.5558 0.2609 1.8915 1.9336 PB2603pax 01.0128 1.4757 1.1082 1.1632 2.3335 0.7457 PB2624pax 0 0.4403 0.51670.7208 1.3259 1.3865 1.2286 PB2824pax 0 0.5178 0.9907 0.4582 1.01321.8135 1.3207 PB2880pax 0 1.0403 1.0617 0.6208 −0.2491 1.8615 1.5586PB3088pax 0 1.6503 1.7517 1.3208 1.1359 3.4165 2.3786 RC0882pax 0 0.27281.4507 0.6582 0.7682 2.0985 1.8007 RC0888pax 0 −0.7872 −0.3193 −0.21180.2932 0.7835 0.2957 RC0968pax 0 −0.8522 −0.6493 0.0182 1.4382 0.78352.1457 RC2114pax 0 −0.4722 0.7557 0.0282 0.4132 0.1335 1.9357 RC2238pax0 0.9528 1.8557 0.5982 1.5182 2.4435 2.8857 RC2681pax 0 0.3278 1.19570.4582 0.5532 0.7435 0.9507 RC2703pax 0 1.6403 1.8317 0.7658 1.19591.1615 1.1436 RC2749pax 0 0.3978 1.1407 0.3632 0.4682 1.6635 1.5157RC2750pax 0 −1.7872 −1.2393 −1.0418 −0.3118 −0.1115 −0.6943 RC2756pax 01.3803 0.9167 0.5508 1.0009 1.6415 1.9636 RC2771pax 0 −0.9547 0.3567−0.6792 −0.3341 1.1965 1.4536 RC2790pax 0 0.8953 0.9817 0.4808 0.72591.0815 1.3536 RC2792pax 0 1.2903 0.8667 0.8058 1.4559 1.9415 1.5836RC2808pax 0 0.7953 1.9117 0.7558 1.0009 1.1715 1.4036 RC2822pax 0 0.77281.2607 0.3282 0.5932 2.5785 0.5807 RC2834pax 0 −0.6247 0.2917 −0.57420.9309 2.0765 0.9236 RC2871pax 0 0.9028 1.4107 1.1032 1.5332 2.64852.6307 RC2879pax 0 −0.2047 0.3867 −0.0342 0.8509 0.6315 0.5086 RC2892pax0 −0.0022 0.5557 −0.0968 0.7032 2.0385 1.1857 RC2895pax 0 1.7178 1.98571.2632 1.3682 1.4435 2.0557 RC2921pax 0 1.3153 1.2967 1.0058 1.37092.0315 2.0886 RC2958pax 0 1.0553 1.1017 0.4958 0.1409 0.4965 1.4836RC3022pax 0 0.3028 0.3007 0.3582 1.0432 1.7635 0.4707 RC3112pax 0 1.25531.4667 0.5908 0.5759 2.5515 1.3286 RC3146pax 0 −0.1572 −0.2793 −0.49180.1432 1.0235 0.8307 RC3184pax 0 2.1353 2.5967 1.0758 1.3559 2.28652.2336 RC3232pax 0 −0.3747 0.5667 −0.5942 0.5309 −0.4385 2.3786RC3324pax 0 0.2128 0.8557 0.0082 0.3282 1.3985 −0.0693 RC3327pax 00.2003 −0.1333 −0.0292 −0.2491 1.2315 1.8086 RC3355pax 0 0.0328 0.5657−0.2668 −0.0418 1.2935 0.4857 RC3380pax 0 −0.4372 0.4907 −0.3518 −0.03180.5235 0.0757 RC3413pax 0 0.6028 1.1907 0.4532 0.0182 0.8435 2.3907RC3421pax 0 −0.0047 0.2917 −0.0142 0.1409 −0.0735 −0.0964 RC3468pax 0−0.1022 −0.0143 −0.0618 −0.3368 1.1485 0.2907 RC3498pax 0 0.0353 −0.2633−0.1892 0.2909 1.2415 0.2486 CC0003pax 1 −1.4122 −0.3593 −0.9318 −0.64680.8435 −0.8893 CD0157pax 1 1.5153 1.5517 1.1308 1.3209 3.0715 2.8186CD0164pax 1 −0.0247 0.4667 0.6758 1.0059 2.5315 1.2886 CD0256pax 1−0.6322 0.0807 0.0032 0.0382 0.8935 1.1557 CD0322pax 1 −1.1572 −0.4693−0.8368 −0.2818 0.5435 1.0507 CD0356pax 1 −0.6772 −1.1393 −0.7718−0.8668 −0.2215 −1.1643 CD0371pax 1 0.1028 0.9607 −0.1918 −0.3018 0.8385−0.2843 CD0629pax 1 −0.0772 0.5407 0.5382 1.3882 0.2885 1.2507 CD1050pax1 0.8153 0.1417 0.2208 0.7559 1.3815 1.0836 DC0001pax 1 0.1353 −0.41830.1208 −0.0941 0.3065 0.0936 DC0002pax 1 0.9878 0.2857 0.3632 −0.17181.7035 0.9057 DS0003pax 1 −1.8347 −0.7183 −1.5192 −1.5591 −0.3485 0.6886FC0005pax 1 0.0928 0.1507 0.2682 0.1732 0.4235 1.8157 FC0011pax 1−0.4572 −0.0793 −0.1118 −0.2318 0.8635 0.2707 FC0012pax 1 −2.5847−0.4033 −1.3792 −1.2541 −0.2885 −0.3614 JGA0001pax 1 −2.3247 −1.6483−1.5592 −0.7241 −1.2535 −1.1814 JGA0008pax 1 −0.6772 1.0357 0.57320.0932 0.8485 0.7057 JH0002pax 1 0.5453 0.9667 0.5608 0.4459 1.40650.7586 JH0003pax 1 0.2853 0.1667 −0.2292 −0.3941 −0.1385 1.0886JH0004pax 1 −0.1747 −0.1733 −0.3592 −0.5591 1.1515 0.6686 JH0005pax 10.0928 1.2207 0.0682 0.1482 1.5735 1.1757 JH0006pax 1 −0.6397 0.0667−0.2692 −0.2741 0.8715 −0.3114 JH0007pax 1 −2.6372 −2.2043 −1.6168−0.5018 −0.0865 −1.2993 JH0008pax 1 0.5453 2.4167 0.2208 0.8859 2.13150.6336 JH0009pax 1 −0.8522 −0.7793 −0.0968 −0.6668 −0.1815 0.4507JH0010pax 1 −0.3847 0.6517 0.2208 0.3059 0.6365 −0.2664 JH0012pax 1−0.1072 0.3857 0.2882 0.0432 0.6685 0.9807 JH0013pax 1 −0.3022 0.2007−0.3118 −0.0568 2.0485 0.8357 JH0014pax 1 1.1828 1.2107 0.3182 −0.04182.0485 2.5207 JH0016pax 1 0.5053 0.9167 0.6658 0.2659 0.4715 0.8636JH0018pax 1 −1.1972 −0.1493 −0.4268 −0.4318 −0.1465 0.3207 JH0019pax 10.1153 0.6267 0.0558 −0.2091 0.1565 0.1936 JH0020pax 1 0.2728 2.02070.7332 −0.1518 0.7585 1.3107 JH0021pax 1 −0.8297 0.7867 −0.3992 −0.85410.2615 0.0586 JH0023pax 1 −0.2272 1.3307 0.5182 1.6882 1.2985 1.9507JH0024pax 1 1.0653 2.5467 1.2508 1.4159 3.3465 2.9786 JH0025pax 1−0.7847 −0.3833 −0.4292 −0.8891 −0.2435 0.5136 JH0026pax 1 −1.15220.5857 −0.0418 −0.3468 1.1035 1.1507 JH0027pax 1 −3.3947 −2.5983 −2.3842−2.0791 −2.3435 −1.8814 JH0028pax 1 0.3253 1.0967 0.9908 0.7459 2.54151.4586 JH0029pax 1 −1.3522 −0.6093 −0.5918 −0.7818 −0.5615 0.5557JH0031pax 1 −0.7297 0.2817 −0.2092 −0.2791 0.5615 0.5136 JH0032pax 11.5478 1.7557 0.4532 0.3732 1.7785 1.8307 JH0033pax 1 −0.8797 1.0817−0.4242 −0.6141 1.3865 −0.1914 JH0034pax 1 −0.5397 1.0667 0.4908 0.39091.5465 1.5186 JH0035pax 1 −0.3222 1.3857 0.2782 −0.3268 1.9385 0.8207JH0036pax 1 0.8128 1.0107 0.4032 0.2182 1.9235 1.1757 JH0038pax 1 0.31031.5367 0.2258 0.4109 2.1215 0.8386 JH0039pax 1 0.5278 0.6207 0.50321.1232 3.1135 0.0857 JH0040pax 1 0.4353 0.8117 0.5508 0.3259 1.24650.8636 JH0041pax 1 0.7303 1.7117 0.4508 0.2759 1.9565 1.0436 JH0042pax 1−2.2922 −0.6293 −1.2268 −0.7568 −1.0515 −1.7443 JH0043pax 1 −0.8022−0.1993 −0.2918 −0.9318 −0.3015 −0.2393 JH0046pax 1 −0.0447 0.05670.6408 −0.2541 0.8965 0.0336 JH0047pax 1 0.1728 1.9907 0.6082 1.27821.6085 2.0757 JH0051pax 1 −0.9647 −0.1533 −0.3892 −1.0491 −0.2135−0.4064 JH0052pax 1 −0.6597 0.3067 −0.4442 −0.9141 −0.4585 −0.7914JH0053pax 1 −0.9522 −0.1693 −0.5918 −0.0318 −0.2515 0.1107 JH0057pax 1−0.1872 1.9757 0.1432 0.6682 1.4335 1.2057 JH0059pax 1 −0.7897 0.0717−0.3442 −0.3241 0.3215 0.1136 JH0060pax 1 −1.0122 0.1207 −0.2818 0.04820.3385 0.3557 JH0061pax 1 0.7803 2.5567 0.5558 1.0309 3.2565 0.3136JH0063pax 1 −0.2197 1.0467 0.2458 −0.4141 0.8115 2.0486 JH0065pax 1−2.7497 −1.4333 −2.0542 −2.8691 −1.5035 −0.4814 JH0066pax 1 −1.1972−0.0343 −0.8418 −1.6318 −1.0715 −0.4643 JH0068pax 1 0.3653 0.8867 0.71080.4809 1.7165 2.1986 JH0069pax 1 −0.6447 0.3967 −0.0892 −0.1791 −0.0735−0.4014 JH0071pax 1 −2.3272 −2.1943 −1.6968 −1.0318 −1.2715 −0.3743JH0072pax 1 −0.1197 −0.7533 −0.0892 −0.6741 1.1515 0.0586 JH0077pax 1−0.3022 0.2957 0.0882 −0.0368 0.4435 1.6307 JH0078pax 1 1.2953 1.27671.2058 0.2609 1.2165 1.2786 JH0080pax 1 −2.1122 −1.1393 −1.0418 −0.7568−1.5315 0.3507 JH0082pax 1 −0.6722 −0.5393 −0.0518 −0.2368 −0.30651.0857 JH0083pax 1 −1.3997 −0.0133 −0.3542 −1.2991 0.3115 0.7236JH0086pax 1 0.2553 0.1667 0.0158 −0.7941 0.4015 1.1086 JH0092pax 1−0.2647 1.6167 0.2158 0.2559 0.1565 1.3386 MH0001pax 1 1.1653 1.77171.0908 1.1159 2.8015 1.9086 MH0009pax 1 −0.4972 −0.0193 −0.7268 −0.5768−0.3015 −0.3493 MH0012pax 1 −0.1347 1.0017 0.3658 0.8559 0.9765 0.9186MH0014pax 1 0.8278 1.4457 0.8482 0.5432 3.0535 1.8007 MH0016pax 1−1.1672 −0.8793 −0.9168 −1.2168 −0.4165 −0.7243 MH0017pax 1 0.34031.4267 0.5658 −0.0241 1.7565 2.0536 MH0018pax 1 1.2128 0.7657 0.32320.3682 2.1235 0.7057 MH0021pax 1 0.8103 0.2517 −0.3192 0.0709 1.68651.7086 MH0022pax 1 0.6903 1.3667 0.6308 0.5459 1.2515 1.4236 MH0024pax 10.2228 0.4757 −0.0568 −0.1768 0.0635 0.9057 MH0026pax 1 −1.2897 0.0417−0.4392 0.1059 −0.3935 −0.1914 MH0028pax 1 −0.0272 0.3557 −0.1068−0.8368 −0.0765 −0.0493 MH0029pax 1 −0.0697 0.0167 0.0658 −0.6641 1.02150.3536 MH0035pax 1 0.6603 2.0217 0.9458 0.7759 1.0965 1.4036 MH0037pax 1−0.1697 1.0717 −0.0342 0.1309 1.1665 1.6186 MH0038pax 1 1.7453 1.59671.3108 1.4609 2.2365 2.3736 MH0039pax 1 −1.1922 −0.3093 −0.1218 −0.5718−0.0965 0.5557 MH0042pax 1 −0.1522 0.0657 0.2332 −0.1318 −0.0815 0.9357MH0050pax 1 −1.4222 0.5507 −0.6818 −0.3468 0.8885 1.6307 MH0051pax 1−1.2197 −1.0683 −0.7092 −1.6041 −2.1735 −0.1814 MIP0004pax 1 −2.1722−2.1793 −1.4118 −1.1218 −0.9865 −2.2593 MP0013Apax 1 0.4028 1.23070.0782 0.2282 1.4535 0.1557 MP0014Bpax 1 −0.4347 0.6117 −0.5542 −0.04411.2765 −0.1964 MP0018Apax 1 −1.0022 −0.4193 −0.9668 −1.5668 −0.3165−1.4293 MP0019Bpax 1 −0.6222 −0.2993 −0.3768 −0.6268 −0.3465 0.8757MP0024pax 1 −1.2597 −0.2333 −0.4542 0.1409 1.3465 0.7386 NK2001pax 1−0.8347 −0.1233 −0.6992 −1.5741 −0.0785 1.1036 NK2002pax 1 0.0203−0.0183 0.1908 −0.4741 0.8115 0.9786 NK2003pax 1 −0.7372 0.0407 −0.3218−0.2868 1.2085 −0.1993 NK2004pax 1 −0.9022 0.3107 −0.5118 −0.9568 0.5285−0.3843 PB1829pax 1 0.3378 1.5057 0.0682 0.0782 1.5485 1.7407 PB1842pax1 1.0953 1.3467 0.8708 0.2009 2.3265 0.9536 PB1872pax 1 0.3928 0.65070.5682 −0.5068 1.2035 0.1807 PB2857pax 1 −0.9422 0.5057 −0.3968 0.2982−0.2165 0.8657 RC2919pax 1 2.0678 2.0357 1.6382 3.0282 2.8985 3.9757RC3062pax 1 0.1453 0.5917 0.1408 0.3759 0.1365 0.3386 RC3277pax 1−0.1122 0.1707 −0.0568 −0.0168 0.7285 0.2457 RC3297pax 1 0.2078 0.94570.4032 0.5332 2.7135 0.9807 RC3445pax 1 −0.8497 −0.1233 −0.3442 −0.73910.6765 0.3086 RC3467pax 1 2.0928 2.9557 1.5782 1.4332 2.8485 2.8457

Surprisingly, analysis of the data showed that RNA encoded by ANXA3,CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1 is present on average at asignificantly higher level (p-value less than 0.05) in blood of subjectshaving colorectal cancer relative to subjects having no colorectalpathology (Table 10). The ranges of fold-change in the levels of RNAencoded by these genes normalized to levels of RNA encoded by IL2RB inblood of the training set subjects having colorectal cancer relative tothe training set subjects not having any colorectal pathology are shownin Table 10.

TABLE 10 Sample training set ranges of fold-change in levels of RNAencoded by ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1 normalized tolevels of RNA encoded by IL2RB in blood of subjects having colorectalcancer relative to subjects not having any colorectal pathology. GeneANXA3 CLEC4D LMNB1 PRRG4 TNFAIP6 VNN1 Average normalized RNA level in−0.30 0.42 −0.05 −0.12 0.79 0.66 subjects having colorectal cancer (ΔCt)Average normalized RNA level in 0.46 0.99 0.39 0.65 1.42 1.25 subjectsnot having any colorectal pathology (ΔCt) Average RNA level fold-change1.69 1.48 1.55 1.35 1.55 1.35 p-value for average RNA level fold-5.5E−09 5.5E−06 6.4E−07 2.5E−13 6.0E−06 2.7E−06 change Maximum observedRNA level 14.43 12.01 6.83 11.46 13.58 11.36 directional fold-change

As can be seen in Table 10, a test subject having a blood level of RNAencoded by ANXA3, normalized to a level of RNA encoded by IL2RB, whichis 1.7 to 14.4 fold higher than the average level of RNA encoded by thisgene in blood of subjects not having any colorectal pathology is morelikely to have colorectal cancer than to not have any colorectalpathology.

As can be seen in Table 10, a test subject having a blood level of RNAencoded by CLEC4D, normalized to a level of RNA encoded by IL2RB, whichis 1.5 to 12.0 fold higher than the average level of RNA encoded by thisgene in blood of subjects not having any colorectal pathology is morelikely to have colorectal cancer than to not have any colorectalpathology.

As can be seen in Table 10, a test subject having a blood level of RNAencoded by LMNB1, normalized to a level of RNA encoded by IL2RB, whichis 1.5 to 6.8 fold higher than the average level of RNA encoded by thisgene in blood of subjects not having any colorectal pathology is morelikely to have colorectal cancer than to not have any colorectalpathology.

As can be seen in Table 10, a test subject having a blood level of RNAencoded by PRRG4, normalized to a level of RNA encoded by IL2RB, whichis 1.3 to 11.5 fold higher than the average level of RNA encoded by thisgene in blood of subjects not having any colorectal pathology is morelikely to have colorectal cancer than to not have any colorectalpathology.

As can be seen in Table 10, a test subject having a blood level of RNAencoded by TNFAIP6, normalized to a level of RNA encoded by IL2RB, whichis 1.5 to 13.6 fold higher than the average level of RNA encoded by thisgene in blood of subjects not having any colorectal pathology is morelikely to have colorectal cancer than to not have any colorectalpathology.

As can be seen in Table 10, a test subject having a blood level of RNAencoded by VNN1, normalized to a level of RNA encoded by IL2RB, which is1.3 to 11.4 fold higher than the average level of RNA encoded by thisgene in blood of subjects not having any colorectal pathology is morelikely to have colorectal cancer than to not have any colorectalpathology.

Generation of Logistic Regression Models for Determining the Probabilitythat a Test Subject has Colorectal Cancer Versus not Having anyColorectal Pathology Via Measurement of Levels of RNA Encoded by ANXA3,CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1 Normalized to Levels of RNAEncoded by IL2RB:

Linear regression analysis of levels of RNA encoded by ANXA3, CLEC4D,LMNB1, PRRG4, TNFAIP6 and VNN1 normalized to IL2RB surprisingly showedthat logistic regression models could be generated, based on bloodexpression levels normalized to IL2RB for all 63 possible combinationsof one or more of these genes, for discriminating, with a ROC AUC of atleast 0.67, between subjects having colorectal cancer and subjects nothaving any colorectal pathology. Examples of these logistic regressionmodels are shown in Table 11. A logistic regression model based onANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1 (Table 11, Model #128) wassurprisingly found to enable discrimination between subjects havingcolorectal cancer and subjects not having any colorectal pathology witha ROC AUC of 0.80.

By way of example, Model #128 of Table 11 corresponds to:

P={1+ê−[(−0.196)+(−1.042)(L _(ANXA3))+(0.393)(L _(CLEC4D))+(1.272)(L_(LMNB1))+(−1.837)(L _(PRRG4))+(0.289)(L _(TNFAIP6))+(−0.153)(L_(VNN1))]}̂−1,

-   -   where P is the probability that a test subject has colorectal        cancer as opposed to not having any colorectal pathology, where        L_(ANXA3) is a ratio of a level of RNA encoded by ANXA3 to a        level of RNA encoded by IL2RB in blood of the test subject,        L_(CLEC4D) is a ratio of a level of RNA encoded by CLEC4D to a        level of RNA encoded by IL2RB in blood of the test subject,        L_(LMNB1) is a ratio of a level of RNA encoded by LMNB1 to a        level of RNA encoded by IL2RB in blood of the test subject,        L_(PRRG4) is a ratio of a level of RNA encoded by PRRG4 to a        level of RNA encoded by IL2RB in blood of the test subject,        L_(TNFAIP6) is a ratio of a level of RNA encoded by TNFAIP6 to a        level of RNA encoded by IL2RB in blood of the test subject, and        L_(VNN1) is a ratio of a level of RNA encoded by VNN1 to a level        of RNA encoded by IL2RB in blood of the test subject.

Further by way of example, Model #157 of Table 11 corresponds to:

P={1+ê−[0.288+(−1.392)(L _(PRRG4))]}̂−1,

-   -   where P is the probability that a test subject has colorectal        cancer as opposed to not having any colorectal pathology, and        L_(PRRG4) is a ratio of a level of RNA encoded by PRRG4 to a        level of RNA encoded by IL2RB in blood of the test subject.

TABLE 11 Logistic regression models based on blood expression levels forany possible combination of one or more of ANXA3, CLEC4D, LMNB1, PRRG4,TNFAIP6 and VNN1, normalized to IL2RB expression levels for determiningthe probability that a test subject has colorectal cancer as opposed tonot having colorectal cancer. ROC AUC values for the models are shownfor the sample training set used to generate the models, as well as foran independent blind sample test set used to test the models. Themodels, listed in order of decreasing ROC AUC value for the trainingset, are based on expression levels determined via quantitative reversetranscriptase-PCR analysis using IL2RB as duplex partner fornormalization. The form of these models is: P = {1 + e{circumflex over( )}−[K₀ + K₁L₁ + K₂L₂ + K₃L₃ . . . + K_(n)L_(n)]}{circumflex over( )}−1, where P is the probability that a test subject has colorectalcancer as opposed to not having any colorectal pathology; K₀ is aconstant; K₁ is a coefficient specific to a first gene; L₁ is a ratio ofa level of RNA encoded by the first gene in blood to a level of RNAencoded by IL2RB in blood; K₂ is a coefficient specific to a secondgene; L₂ is a ratio of a level of RNA encoded by the second gene inblood to a level of RNA encoded by IL2RB in blood; K₃ is a coefficientspecific to a third gene; L₃ is a ratio of a level of RNA encoded by thethird gene in blood to a level of RNA encoded by IL2RB in blood; K_(n)is a coefficient specific to an nth gene; and L_(n) is a ratio of alevel of RNA encoded by the nth gene in blood to a level of RNA encodedby IL2RB in blood. No regression coefficients are specified for geneswhich are not included in the gene combination (indicated by “—”) onwhich a given logistic regression model is based. No. of Logistic genesROC AUC Gene-specific regression coefficient Regression in TrainingConstant (K_(n)) Model # Model Set Test Set (K₀) ANXA3 CLEC4D LMNB1PRRG4 TNFAIP6 VNN1 128 6 0.80 0.78 −0.196 −1.042 0.393 1.272 −1.837  0.289 −0.153 129 5 0.80 0.79 −0.298 −1.058 0.366 1.187 −1.854   0.285— 130 5 0.80 0.79 0.034 −0.945 0.456 1.300 −1.715 — −0.146 131 5 0.790.78 −0.070 −0.937 — 1.469 −1.774   0.336 −0.115 132 4 0.79 0.79 −0.065−0.961 0.428 1.221 −1.733 — — 133 4 0.79 0.78 −0.154 −0.955 — 1.394−1.790   0.330 — 134 5 0.79 0.78 −0.305 −0.630 0.575 — −1.632   0.311−0.049 135 4 0.79 0.78 0.229 −0.799 — 1.537 −1.620 — −0.097 136 4 0.790.79 −0.337 −0.645 0.562 — −1.642   0.309 — 137 4 0.79 0.78 −0.058−0.516 0.645 — −1.493 — −0.041 138 5 0.79 0.76 0.239 — 0.173 0.355−1.782   0.144 −0.210 139 3 0.79 0.78 0.153 −0.817 — 1.474 −1.637 — —140 3 0.79 0.79 −0.085 −0.528 0.634 — −1.503 — — 141 4 0.79 0.76 0.128 —0.280 — −1.707   0.174 −0.162 142 4 0.79 0.76 0.337 — 0.216 0.413 −1.721— −0.203 143 3 0.79 0.77 0.229 — 0.355 — −1.615 — −0.144 144 4 0.79 0.760.279 — — 0.492 −1.757   0.172 −0.189 145 4 0.78 0.76 0.109 — 0.1280.225 −1.802   0.134 — 146 3 0.78 0.76 0.053 — 0.209 — −1.747   0.157 —147 3 0.78 0.76 0.416 — — 0.607 −1.673 — −0.173 148 3 0.78 0.76 0.206 —0.169 0.285 −1.745 — — 149 3 0.78 0.76 0.150 — — 0.339 −1.781   0.156 —150 2 0.78 0.77 0.153 — 0.285 — −1.659 — — 151 2 0.78 0.76 0.284 — —0.457 −1.703 — — 152 3 0.78 0.76 0.123 — — — −1.591   0.257 −0.078 153 30.78 0.77 −0.102 −0.352 — — −1.463   0.390 — 154 4 0.78 0.77 −0.130−0.368 — — −1.474   0.388   0.037 155 2 0.78 0.76 0.082 — — — −1.630  0.237 — 156 3 0.77 0.77 0.216 −0.179 — — −1.276 —   0.061 157 1 0.770.76 0.288 — — — −1.392 — — 158 2 0.77 0.77 0.267 −0.150 — — −1.256 — —159 2 0.77 0.76 0.296 — — — −1.384 — −0.011 160 5 0.73 0.75 0.162 −0.9070.153 0.321 — −0.073 −0.270 161 4 0.73 0.75 0.108 −0.930 0.130 0.296 — —−0.273 162 3 0.73 0.76 0.086 −0.818 0.185 — — — −0.243 163 4 0.73 0.760.125 −0.793 0.206 — — −0.055 −0.238 164 4 0.72 0.75 0.208 −0.868 —0.406 — −0.046 −0.254 165 3 0.72 0.75 0.167 −0.888 — 0.380 — — −0.258166 2 0.72 0.75 0.171 −0.698 — — — — −0.204 167 3 0.72 0.75 0.179 −0.691— — — −0.009 −0.203 168 3 0.72 0.76 −0.021 −0.870 0.134 — — −0.077 — 1694 0.72 0.76 −0.012 −0.929 0.103 0.155 — −0.087 — 170 2 0.72 0.77 −0.080−0.907 0.102 — — — — 171 1 0.72 0.76 −0.014 −0.827 — — — — — 172 2 0.720.76 0.031 −0.793 — — — −0.043 — 173 3 0.72 0.76 −0.079 −0.957 0.0740.122 — — — 174 2 0.72 0.76 −0.039 −0.932 — 0.178 — — — 175 3 0.72 0.760.026 −0.902 — 0.221 — −0.068 — 176 3 0.71 0.72 0.514 — — −0.502   —−0.191 −0.309 177 4 0.71 0.72 0.519 — −0.024   −0.482   — −0.186 −0.306178 3 0.70 0.72 0.398 — −0.097   −0.604   — — −0.319 179 2 0.70 0.730.361 — — −0.706   — — −0.333 180 2 0.70 0.70 0.727 — — — — −0.345−0.451 181 3 0.70 0.71 0.701 — −0.192   — — −0.256 −0.383 182 2 0.700.73 0.306 — — −0.769   — −0.227 — 183 3 0.69 0.73 0.331 — −0.085  −0.690   — −0.208 — 184 1 0.69 0.73 0.102 — — −1.041   — — — 185 2 0.690.70 0.589 — −0.367   — — — −0.433 186 2 0.69 0.73 0.186 — −0.170  −0.838   — — — 187 1 0.68 0.69 0.552 — — — — — −0.671 188 2 0.68 0.720.548 — −0.379   — — −0.331 — 189 1 0.67 0.70 0.549 — — — — −0.576 — 1901 0.67 0.71 0.371 — −0.649   — — — —

Blind Sample Test Set:

Quantitative reverse transcriptase-PCR analysis of expression of ANXA3,CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1 in an independent test set ofblood samples from 165 subjects having colorectal cancer and 171subjects not having any colorectal pathology was performed as describedabove for the training set. The normalized RNA levels measured are shownin Table 12.

TABLE 12 Sample test set levels of RNA encoded by ANXA3, CLEC4D, LMNB1,PRRG4, TNFAIP6 and VNN1 in blood of subjects having colorectal cancer(Group 1) and subjects not having any colorectal pathology (Group 0),normalized to levels of RNA encoded by IL2RB. Levels shown correspond toΔCt. Gene Sample ID Group ANXA3 CLEC4D LMNB1 PRRG4 TNFAIP6 VNN1CD0036pax 0 −0.0922 −0.0443 −0.1868 −1.0168 0.5035 −0.0543 CD0053pax 01.1828 2.1407 1.5032 1.3032 2.9135 1.5907 CD0092pax 0 1.2028 0.90570.8232 0.6032 1.6285 0.2907 CD0108pax 0 −0.0522 −0.1893 0.2582 −0.13180.3385 0.2757 CD0122pax 0 0.5628 1.2857 0.5582 0.6382 2.0485 1.9357CD0148pax 0 0.6778 2.0057 0.6732 1.4432 2.0335 1.5607 CD0192pax 0 0.68780.8157 0.7682 1.5882 0.7085 0.8907 CD0204pax 0 0.1978 0.6907 0.0782−0.1418 1.0185 0.9557 CD0214pax 0 0.4478 0.1557 1.1682 1.1932 0.62350.6157 CD0237pax 0 0.3828 0.8707 −0.0168 0.0582 1.1685 0.8007 CD0238pax0 0.2428 0.8957 0.3982 1.0232 1.5385 0.9657 CD0242pax 0 0.9528 1.39570.5082 −0.3818 0.9685 1.0307 CD0244pax 0 0.4928 0.3507 0.4182 0.89821.4535 −0.0493 CD0277pax 0 0.1453 1.7617 0.2358 0.4909 1.8265 0.1486CD0282pax 0 −0.1572 0.2207 −0.2118 0.0632 0.7585 −1.2243 CD0295pax 00.1578 0.7957 0.5482 0.5332 1.1735 0.1907 CD0354pax 0 0.5178 0.95570.2432 1.0732 1.6235 1.0307 CD0367pax 0 0.7778 0.6857 0.5232 0.34321.8935 0.8457 CD0369pax 0 1.0328 2.1057 0.9982 1.9532 2.1885 1.7607CD0398pax 0 2.1128 2.2157 1.6682 2.0432 3.7635 3.1407 CD0409pax 0−0.2272 0.7757 0.0282 0.7082 0.5735 1.2357 CD0419pax 0 0.2503 0.96170.5758 0.3759 2.1815 −0.3114 CD0432pax 0 0.3628 0.5607 0.2532 0.02822.0485 0.4407 CD0437pax 0 −0.8872 −0.1793 −0.6918 −0.7768 0.4635 −0.1593CD0472pax 0 −2.1022 −0.4543 −1.0718 −0.6968 0.0235 −0.4243 CD0482pax 03.2778 3.4857 2.2282 2.2532 4.6085 3.4507 CD0484pax 0 0.2778 1.19070.3932 1.3182 1.7635 0.4107 CD0507pax 0 1.4228 1.0757 1.0982 0.42822.4985 1.5357 CD0547pax 0 −1.6622 −0.1543 −0.5068 0.0632 0.1035 −0.3643CD0571pax 0 0.9378 1.2407 1.0382 1.8282 2.3535 0.5757 CD0580pax 0 1.29781.6557 0.5582 0.4382 2.3585 1.2807 CD0583pax 0 −0.4472 −0.9493 −0.4968−0.5218 0.7935 −0.6093 CD0603pax 0 0.6928 0.5507 0.1582 −0.0818 1.41351.2457 CD0604pax 0 0.0028 0.6557 −0.1218 −0.1268 1.6635 −0.1443CD0619pax 0 −0.3072 0.2557 0.1532 0.5182 0.3285 0.8407 CD0637pax 00.3428 0.0357 −0.0618 0.3132 0.8935 0.6657 CD0667pax 0 −0.5672 −0.4993−0.5168 −0.1568 0.0235 −0.2643 CD0670pax 0 3.0478 2.9307 1.7582 1.58823.3985 2.6957 CD0676pax 0 −0.7022 −0.6343 −0.5518 −1.0468 0.2485 0.0357CD0687pax 0 1.4528 2.5807 1.6032 1.9382 1.9035 2.4357 CD0715pax 0−2.1372 −1.5893 −1.0468 −0.7768 −0.3665 0.4757 CD0721pax 0 0.1778 1.18070.0382 0.0582 1.1935 0.7607 CD0726pax 0 −0.2472 −0.2193 0.0432 −0.03180.8985 −1.1393 CD0743pax 0 0.4178 −0.4243 0.2032 0.2782 1.6735 −0.0243CD0786pax 0 0.6678 1.0807 0.6132 0.9682 1.8735 1.0607 CD0800pax 0 2.64782.7757 1.9282 2.4682 3.8585 3.0107 CD0829pax 0 −0.0722 0.4757 0.2082−0.3318 0.5785 0.7957 CD0833pax 0 0.1378 −0.3693 0.0982 0.5032 0.8185−0.2293 CD0840pax 0 −0.5072 0.4157 −0.2018 −0.5618 0.6035 0.1307CD0843pax 0 0.2853 −0.1433 −0.2192 −0.5291 0.3415 0.2136 CD0937pax 01.2178 0.8507 0.9632 1.4432 2.6285 1.8457 CD1001pax 0 0.7553 0.83670.2758 −0.3541 2.7865 0.9686 CD1032pax 0 −3.0422 −2.3193 −0.7918 −0.7218−1.1065 0.0557 CD1068pax 0 −0.1722 −0.0343 0.1382 0.5532 2.0935 −0.2243CD1134pax 0 0.4253 1.2867 0.2608 0.4409 1.3565 1.9236 CD1269pax 0 0.03781.3507 0.1732 −0.2218 1.1735 1.1007 CD1270pax 0 0.8328 0.8557 0.64820.3532 1.4535 1.4157 CD1271pax 0 1.6778 2.4257 1.2282 0.6282 0.98851.8207 CD1278pax 0 0.2578 1.0757 0.5232 0.1582 1.2685 1.3507 CD1285pax 00.6903 0.3917 0.8458 0.3609 2.7765 0.8986 CD1313pax 0 1.7278 1.37570.8182 1.0282 2.8285 0.6057 CD1320pax 0 −0.8972 0.2757 −0.6068 −0.06680.1335 0.7907 CD1329pax 0 0.1903 −0.0983 0.2308 −0.3091 1.1115 0.8936CD1349pax 0 −0.1622 0.5757 −0.3668 −0.9268 0.3935 0.5807 CD1401pax 0−0.0397 −0.2933 −0.2492 0.2409 0.2715 0.2036 CD1428pax 0 2.0228 2.12571.1632 2.0382 2.5385 1.4807 CD1438pax 0 −0.1572 0.3757 −0.1418 0.37820.6935 −0.1493 CD1441pax 0 −0.6972 0.3307 0.0582 0.1132 0.7235 −0.3693CD1458pax 0 0.2328 0.2407 0.9082 0.7332 1.8935 0.5707 CD1487pax 0 0.23781.9757 0.9582 1.6032 1.9785 0.5407 CD1559pax 0 −0.0022 0.8557 0.16320.3532 1.8685 1.0657 CD1561pax 0 1.1728 0.7557 0.6132 0.5432 1.86351.9307 CD1567pax 0 0.6928 1.4807 0.4982 1.2232 1.6035 1.2807 CD1627pax 0−1.0072 −0.6693 −0.1868 0.2632 −0.4965 −0.2743 CD1708pax 0 1.3903 1.84171.1458 0.6909 2.4365 1.8636 CD1719pax 0 −0.4022 0.0407 −0.3068 −0.75680.1285 0.4657 CD1728pax 0 1.8578 2.0957 1.2732 2.0332 1.9635 3.0907CD1741pax 0 −0.4922 −0.5493 −0.1868 −0.4618 1.0035 0.3257 PB0662pax 00.0003 0.3867 0.6908 0.6809 1.2815 −0.1464 PB0701pax 0 0.8803 1.50171.2058 2.1159 1.6765 3.1036 PB0790pax 0 0.4253 0.2867 0.5158 0.54590.1665 −0.2414 PB1222pax 0 −0.0272 0.4407 0.2932 −0.6868 −0.0465 0.8957PB1260pax 0 −1.3447 −1.3983 −0.3742 −1.2441 −0.6585 −0.5814 PB1275pax 0−0.7447 0.3767 −0.3092 0.2359 −0.1835 2.1786 PB1324pax 0 −0.1597 0.56670.4508 1.1109 1.7015 1.8886 PB1336pax 0 0.4853 0.6817 0.2158 −0.59911.1365 1.5136 PB1446pax 0 −0.9897 −0.0133 −0.1992 0.0159 0.7765 1.0086PB1514pax 0 −1.2222 −0.2093 −0.3418 −0.4868 0.8185 −0.0293 PB1540pax 0−0.7672 −0.2493 −0.2668 −0.0068 0.5185 0.8157 PB1700pax 0 0.5403 0.75170.5258 0.8759 1.3265 0.8836 PB1763pax 0 2.0953 2.5067 1.7958 2.64093.0115 1.9736 PB1785pax 0 0.9903 1.6717 0.6008 1.4309 1.3415 0.9486PB1871pax 0 0.4853 0.3717 0.2008 0.3059 1.3365 1.4286 PB1918pax 0 2.66782.3457 1.4632 1.7882 3.8835 2.6107 PB1944pax 0 0.0028 −0.4843 0.44320.7482 0.5435 1.3507 PB1952pax 0 1.9703 1.9167 0.5508 1.8859 2.98652.0386 PB1973pax 0 2.7753 2.8667 1.9308 3.1009 3.7065 3.7736 PB1984pax 0−2.2397 −1.7683 −1.8492 −1.7341 −1.4835 −1.3114 PB2005pax 0 1.48031.5917 0.8408 1.1159 3.3915 1.8386 PB2015pax 0 2.4703 1.9567 2.09083.2409 4.2865 2.5086 PB2024pax 0 0.8053 1.3967 0.8908 1.1359 2.65152.2286 PB2041pax 0 −0.5647 0.7517 −0.0692 0.1909 1.7565 0.1836 PB2062pax0 2.4703 2.4517 1.7908 2.2809 3.3065 2.6786 PB2084pax 0 1.3553 1.05170.6808 1.5959 2.3965 1.4886 PB2130pax 0 0.4353 0.6717 0.0408 −0.10910.6315 0.6436 PB2179pax 0 0.6453 1.5017 0.5358 0.3559 0.7115 0.8686PB2184pax 0 −0.8047 −0.1983 −0.3292 0.0859 0.2715 1.4886 PB2258pax 0−0.0397 −0.9183 −0.5042 −0.4191 0.8415 −1.1164 PB2272pax 0 2.2653 2.27171.6408 1.7259 4.0015 1.8136 PB2342pax 0 2.9678 2.7557 1.1682 1.67322.4335 2.7607 PB2464pax 0 1.0003 1.6717 0.8458 0.4759 1.4465 3.2686PB2516pax 0 0.1603 0.8067 0.4858 0.2109 0.6065 1.9036 PB2564pax 0 0.12531.3217 0.0258 0.3009 0.5815 0.7836 PB2634pax 0 0.0153 1.1567 0.89580.7009 2.5465 0.5586 PB2682pax 0 −1.2297 −0.1383 −0.5242 −1.4441 0.0415−0.0114 PB2709pax 0 1.2978 1.7457 1.3832 1.9982 2.2535 1.3107 PB2711pax0 −0.8797 −1.1033 −0.3892 −0.8391 0.5215 0.4736 PB2757pax 0 0.53531.6117 0.8508 0.6659 1.5865 1.9836 PB2809pax 0 −0.1722 0.9957 0.0032−0.1768 0.6335 0.9057 PB2842pax 0 −0.5547 0.3867 −0.3492 0.0209 −0.25850.3486 PB2875pax 0 −0.0497 1.2967 0.3958 0.3959 1.2365 1.1736 PB2889pax0 0.6453 1.9717 1.0808 1.4209 1.4015 1.5286 PB2909pax 0 −0.1372 −1.0543−0.4368 −0.7268 0.2735 −0.3993 PB2924pax 0 0.7803 1.5167 1.1858 1.21591.3115 1.2786 PB2927pax 0 2.1453 0.8717 0.7158 0.1309 1.5815 2.1286PB2931pax 0 0.2203 −0.3283 −0.8242 −0.8241 1.8315 −0.3664 PB2951pax 0−0.0897 −1.6683 −1.0792 −0.2891 −0.3035 −0.9164 PB2974pax 0 −0.25470.8417 0.1958 0.2259 1.0515 2.2086 PB2978pax 0 3.1253 3.2667 2.24582.4059 3.7165 2.9886 PB2988pax 0 −1.5847 −1.3333 −0.7892 −1.1941 −0.2435−0.3764 PB3014pax 0 1.3953 1.9017 1.2458 0.7959 2.3615 2.5236 PB3021pax0 0.3053 2.1667 0.4108 0.9459 0.9015 0.7786 PB3032pax 0 −0.1547 0.48670.6658 0.0409 1.5065 1.1036 PB3163pax 0 0.3003 1.4117 0.5908 1.14592.4115 1.7336 PB3193pax 0 −0.0047 0.3717 0.0058 0.4559 1.1065 0.7036PB3200pax 0 0.2653 1.0967 0.8608 1.0859 1.4265 1.5786 PB3226pax 0 0.32030.9467 0.4658 0.6709 1.9165 1.1536 PB3227pax 0 −0.0147 0.7317 0.46580.6559 2.0115 1.3936 PB3361pax 0 −0.8197 −0.1133 −0.5592 −0.9891 0.8715−0.6564 PB3439pax 0 −1.2272 −1.8643 −1.1768 −1.2568 −0.9015 −1.7543PB3445pax 0 1.6203 2.4017 1.5208 2.2859 2.7265 2.2836 PB3481pax 0−0.0397 0.9167 0.3308 1.1359 0.5015 1.3636 PB3513pax 0 −0.0347 −0.2133−0.5142 −0.6891 0.8765 −0.9214 PB3524pax 0 0.2103 −0.5733 0.1758 0.18591.2865 0.8486 PB3533pax 0 −0.3047 −0.7483 −0.2442 −0.0641 1.0465 −0.2664PB3568pax 0 1.3153 1.4267 0.9858 1.0159 1.8915 0.8386 PB3582pax 0 1.47032.3517 1.7258 1.8509 3.3165 2.4236 PB3594pax 0 1.0753 1.2267 0.69581.2059 2.4615 1.2786 PB3806pax 0 −1.7122 0.1507 −0.6168 −0.1418 −0.2615−0.3843 PB3828pax 0 −0.8197 0.3417 −0.2942 −0.6091 −0.2085 0.4136PB3863pax 0 1.9153 1.8567 1.5808 1.4659 2.7065 2.5436 PB3877pax 0 2.10032.3217 1.6458 1.3909 3.4115 2.2186 RC2112pax 0 1.0653 2.1567 1.30081.7109 1.6115 2.0886 RC2236pax 0 1.1903 1.2017 1.0508 1.2159 1.51652.2136 RC2239pax 0 −0.2897 1.0767 1.0608 1.3759 0.5315 1.6386 RC2252pax0 2.5353 1.9417 1.4658 1.9359 1.9515 2.7336 RC2338pax 0 1.1953 2.11671.1358 1.0609 2.2215 2.0636 RC2565pax 0 0.0753 1.1417 0.0458 1.26590.5465 2.0486 RC2615pax 0 −0.4847 0.1417 0.3558 0.5509 0.2765 1.1436RC2699pax 0 1.1453 1.5167 0.8658 0.6759 1.7615 1.5636 RC2716pax 0 0.27030.4317 0.7308 1.1609 2.2865 0.9336 RC2728pax 0 −0.1697 1.2267 0.81080.5209 1.1265 0.4386 RC2768pax 0 1.2553 1.6867 0.9908 1.5259 1.88652.7936 RC2782pax 0 2.7703 3.4667 2.2058 2.3409 5.0215 2.7836 RC2869pax 01.3178 1.1907 0.7582 1.3432 2.3085 2.1657 RC2897pax 0 0.1853 0.7067−0.0642 0.3409 1.1865 1.0436 RC2986pax 0 0.8953 0.9867 0.3458 0.43592.5115 1.4186 RC3191pax 0 1.4403 2.2267 1.2558 1.0409 1.9615 2.2036RC3214pax 0 0.7903 1.6817 0.8258 0.8409 2.5365 1.1586 RC3379pax 0 1.30031.7467 1.0158 1.0509 2.4615 0.5686 RC3420pax 0 0.9153 1.0217 0.92581.6209 2.5815 0.4436 AN0001pax 1 −2.1122 −1.1143 −1.0218 −1.6018 −0.1515−0.4493 AN0003pax 1 −3.9172 −2.1693 −2.9568 −1.2268 −2.2065 −2.5393AN0007pax 1 −3.0572 −1.7593 −2.1818 −3.1218 −2.5615 −1.0493 AN0009pax 1−0.3772 0.6607 0.0832 −0.0718 1.9985 1.0707 AN0012pax 1 0.5478 1.12070.3632 −0.0868 1.6385 1.6507 AN0013pax 1 0.2178 2.0007 0.9732 0.13322.7685 2.1157 AN0020pax 1 −1.8472 −0.4843 −1.1768 −1.7718 −1.3165−0.3293 AN4011pax 1 −0.0972 0.8507 0.9782 0.5082 2.1785 0.9257 AN4012pax1 1.6828 1.2857 1.1782 1.2582 2.7935 2.4957 AN4013pax 1 0.7928 2.00570.8132 0.6482 1.6385 1.8907 AN4014pax 1 −1.0472 0.2207 −0.1918 0.12320.4685 1.9757 AN4017pax 1 1.0003 1.3817 0.3358 0.2709 0.4565 1.2836BE3001pax 1 −1.4472 0.2507 −0.3068 −0.6768 −0.8115 0.8657 CC0001pax 1−0.2222 0.1107 −0.1668 −0.0168 1.2635 0.7907 CC0002pax 1 −0.5572 −0.8993−0.6118 −1.9568 −0.2715 0.0957 CC0004pax 1 −1.2622 −1.0993 −0.4268−0.1268 0.5335 0.6457 CC0005pax 1 0.8628 1.2457 0.6532 0.9132 1.82850.4607 CC0006pax 1 0.3128 0.4957 0.2332 0.5682 1.7235 2.2507 CC0007pax 1−1.5122 0.0857 −0.2318 0.0032 −0.1365 0.3407 CC2001pax 1 −0.6672 1.0957−0.0268 0.1382 0.9435 0.7757 CC2002pax 1 −0.6022 −0.9543 −0.6518 −1.5068−0.2315 −0.3993 CD1111pax 1 1.5528 0.9857 1.1082 1.6382 2.0285 2.2057CD1260pax 1 −0.4922 −0.5393 0.0532 −0.5368 1.1035 0.7257 CD1351pax 10.3978 0.4007 −0.0268 −0.3368 1.6485 0.5707 CD1571pax 1 0.6253 0.77170.9908 1.7909 2.5465 1.3386 CD1690pax 1 0.4278 0.5007 0.4682 0.52321.3985 −0.1993 DC0003pax 1 0.3378 0.8707 0.1182 0.0432 0.8135 1.8807DC0005pax 1 0.6328 1.6957 0.6782 0.4432 1.5635 1.7007 DC0008pax 1 0.36280.7757 0.4632 −0.3718 1.6735 0.2907 DC0011pax 1 −0.1322 0.4157 0.55320.5082 1.5035 1.6757 DC0012pax 1 −1.2222 −0.4443 −0.6868 0.3132 −1.1115−0.2343 DC1002pax 1 −1.2322 −0.7593 −0.3968 −0.3968 0.5535 0.3907DC2005pax 1 −2.3922 −1.8993 −1.9468 −0.6968 −1.5515 −0.4343 DC2006pax 10.1778 0.4107 0.1982 −0.6518 1.0185 0.8557 DC3003pax 1 −0.2272 0.95070.1682 −0.4168 1.5985 1.8207 DC5006Apax 1 −0.0922 0.2057 0.2232 −0.3768−0.1115 0.2157 DC5008Apax 1 −0.5972 −0.1043 −0.7018 −0.5168 0.83351.1557 DES1001pax 1 −1.8422 −0.8843 −1.1918 −1.4518 0.8185 −0.4143DES1002pax 1 −0.6822 −0.8993 −0.0418 −0.5318 1.1235 −0.7943 JH0022pax 10.0828 0.5507 0.4282 0.5332 0.5935 1.0307 JH0076pax 1 −0.4872 −0.7793−0.2418 −1.6768 −0.1715 0.1407 JH0085pax 1 −1.0822 −0.6293 −0.4668−1.2268 −0.0365 −0.0193 JH0089pax 1 −0.1597 1.0267 −0.1642 −0.56910.7915 1.1336 JH0090pax 1 −0.7322 −1.0293 −0.4218 −0.0518 0.0435 0.4657JH0091pax 1 0.1353 0.2617 0.2708 1.2709 1.2965 1.1536 JH0093pax 1 0.24281.4557 0.4232 −0.5368 1.5685 0.9107 JH0096pax 1 −2.0347 −0.6183 −1.4842−0.6891 −2.2235 0.8736 JH0097pax 1 −0.4422 −0.5293 −0.0218 −0.79680.4985 −0.7193 JH0100pax 1 0.3803 1.2117 0.4858 0.6959 1.7015 1.0036JH0101pax 1 0.8128 0.4507 0.6232 0.3482 1.6835 0.5857 JH0105pax 1 0.38781.1657 0.3682 −0.5768 1.7485 2.0007 JH0106pax 1 −0.6497 −0.2083 0.0308−0.1191 0.8115 1.1536 JH0108pax 1 −0.0772 0.6557 0.3282 −0.0768 0.84851.1157 JH0109pax 1 0.9803 1.9517 0.7158 0.2559 1.7615 2.2986 JH0110pax 1−0.5072 0.2307 −0.1668 −0.6018 −0.3515 0.7807 JH0111pax 1 0.7503 1.77170.4808 1.8809 2.4715 1.1486 JH0113pax 1 −0.3047 0.5867 0.3308 −0.84410.5715 0.1886 JH0114pax 1 −2.0472 −1.6493 −1.3618 −0.6518 −1.7715−2.1743 JH0116pax 1 −0.2772 1.1207 0.2182 0.6082 0.7885 0.7457 JH0117pax1 −1.6972 −0.6243 −1.0418 −0.8018 −0.1765 0.4257 JH0118pax 1 0.3503−0.0433 0.5258 −0.0441 1.1065 0.4236 JH0120pax 1 −0.3797 0.2467 −0.0792−1.2341 1.4265 0.0686 JH0123pax 1 −1.8597 −0.9733 −0.7492 −0.5891−0.8185 −1.2464 JH0126pax 1 −1.7822 −0.3243 −0.7168 −1.3618 −0.66650.2307 JH0127pax 1 −0.3897 1.0367 0.4308 0.7759 0.6415 0.6086 JH0129pax1 0.3903 1.2817 0.6808 1.0259 2.6865 1.8886 JH0130pax 1 −0.9022 −0.3543−0.7818 −0.9818 −0.4465 0.2657 JH0131pax 1 −0.4922 −0.5243 −0.5968−1.3968 −0.8715 0.2057 JH0132pax 1 −0.3897 0.1117 −0.0742 0.3959 0.46150.1436 JH0135pax 1 −1.3622 −1.6143 −1.2368 −1.1018 −1.2465 −1.4743JH0136pax 1 −2.6172 −1.9043 −1.3118 −1.4768 −1.1965 −2.0743 JH0137pax 1−0.1372 0.9407 −0.3768 −0.6168 0.7685 0.5507 JH0138pax 1 −0.5622 0.19570.0532 −0.3068 0.2285 0.2757 JH0139pax 1 −0.7247 −0.6583 −0.5792 −1.1191−0.4935 −0.8464 JH0142pax 1 −0.4547 0.7017 0.3608 0.4459 0.7815 −0.1964JH0144pax 1 0.3128 2.3157 0.8682 −0.1368 2.0135 1.2707 JH0147pax 10.3778 1.4257 0.4532 0.5232 2.9535 0.7407 JH0149pax 1 0.5528 1.24070.3682 0.1432 2.5535 0.9357 KW0002pax 1 −0.4072 0.4307 −0.1868 −0.49680.6785 1.3907 KW0003pax 1 0.0253 1.3217 0.7908 −0.0691 0.1415 3.0886MH0053pax 1 2.2253 2.3967 1.2558 0.6209 3.2815 2.1886 MH0057pax 1−0.6947 −0.5333 −0.4792 −0.5041 0.5915 −0.5264 MH0059pax 1 −2.8497−2.1083 −2.1892 −1.5691 −0.3235 −1.1764 MH0062pax 1 −0.9697 −0.1633−0.0592 −0.4891 0.5065 0.3186 MH0065pax 1 −1.7097 −1.6233 −0.8042−0.9491 −0.6235 −1.1314 MH0066pax 1 −1.4297 −0.9733 −1.0642 −0.8041−0.5235 −0.9314 MH0068pax 1 2.4828 2.4407 1.7882 1.6982 3.0035 1.0707MH0070pax 1 0.1703 0.8467 0.5808 0.3859 1.4015 1.1036 MH0073pax 1−1.5622 −0.8493 −1.3868 −1.4018 −0.1715 −0.7993 MH0074pax 1 −1.0422−1.1393 −0.6218 −0.6218 0.7535 −1.2193 MH0076pax 1 0.2653 1.8167 0.0658−0.4241 2.0165 2.0386 MH0077pax 1 −0.2772 0.3357 −0.2418 −0.6218 0.29350.4157 MH0078pax 1 −1.2497 −0.0583 −0.6992 −1.7091 −0.7685 1.0236MH0079pax 1 0.6153 0.7767 0.5608 −0.0241 0.6065 1.1786 MH0080pax 1−0.1597 0.2867 0.5258 0.3709 1.5765 0.6186 MH0081pax 1 0.1403 1.23670.1658 −1.1941 1.7615 0.7686 MH0082pax 1 0.8203 1.4817 0.4558 −0.14411.6365 0.7336 MH0083pax 1 1.5778 1.7007 0.9332 1.2532 2.2185 1.6807MH0087pax 1 −0.9922 0.9707 −0.0668 0.5032 1.4985 0.6507 MH0088pax 1−0.2172 0.8457 0.3582 −0.1868 0.8535 0.5157 MH0089pax 1 −2.2197 −1.5133−1.0292 −1.5141 −0.8685 −0.3364 MH0090pax 1 −0.9247 0.3217 −0.2792−0.6841 0.0065 −0.4964 MH0095pax 1 −2.1147 −1.5633 −1.4142 −1.8041−1.0985 −0.9514 MIP0002pax 1 −1.8047 −0.3533 −0.6842 −0.1141 −0.06850.4836 MIP0003pax 1 0.0778 0.1157 −0.1818 0.0282 0.7585 −0.1643MIP0005pax 1 −2.2797 −1.3883 −1.6292 −1.1741 −0.8035 −1.7414 MIP0008pax1 0.5053 0.8367 0.0008 −0.2191 1.8415 0.7086 MIP0009pax 1 −1.3122−1.1793 −1.2268 −1.4768 −0.6215 −1.0043 MIP1007pax 1 −1.1547 −0.4633−0.4792 0.1859 0.2515 −1.4864 MIP1009pax 1 −2.9997 −1.6083 −2.0442−1.4391 −0.7485 −1.1164 MIP1011pax 1 −0.6497 −0.7283 −0.8992 −1.04910.3015 −0.8914 MIP1013pax 1 −0.0222 0.8707 0.0282 0.0432 0.7335 1.4007MIP2002pax 1 0.8603 1.4317 0.7358 0.4159 1.6165 1.3386 MIP2003pax 1−0.2947 0.3567 0.2508 0.6959 1.8515 0.8486 MIP2006pax 1 −0.3622 0.74570.0432 0.5432 0.8035 0.3157 MIP3003pax 1 −2.5772 −1.5843 −2.1418 −1.4368−1.7415 −1.3193 MIP3004pax 1 −0.9222 −0.5393 −0.4618 −0.3918 0.6435−0.6943 NK1001pax 1 0.4828 1.0657 0.0782 0.1832 1.9635 0.9307 NK1003pax1 −0.9747 0.1017 −0.1192 −0.3691 0.4365 0.4536 NK1004pax 1 −0.7947−0.5133 −0.4642 −0.8441 0.6715 0.0186 NK1005pax 1 −0.1597 1.5217 0.80580.4809 2.0815 0.9936 NK1008pax 1 −1.6222 0.1407 −0.5918 −0.2818 −0.02150.2257 NK1009pax 1 1.6828 1.5207 0.7182 0.4582 2.3135 1.0907 NK2005pax 1−1.5347 −0.9483 −0.6192 −0.8041 −1.9435 −0.0114 NK2006pax 1 −1.9272−1.2543 −1.7168 −1.1568 −0.7815 −1.6093 NK2007pax 1 0.1978 0.4957 0.12820.6782 1.0285 −0.0693 NK2008pax 1 −1.9022 −1.2193 −1.0068 −1.3468−0.0615 −1.4393 NK2009pax 1 −0.4097 0.3167 0.0658 −0.7691 1.5165 −0.0614NK2010pax 1 −1.1147 −0.5983 −0.4692 −0.8091 −0.3935 −0.7364 NK2014pax 10.6753 1.6317 1.0258 1.0759 2.0515 1.1686 NK2015pax 1 −0.6222 0.63070.0482 −0.4318 1.6935 1.3107 NK2016pax 1 −0.4297 0.2167 −0.2092 0.06591.6815 0.4936 NK2018pax 1 −1.9522 −1.1093 −0.8468 −0.9018 −0.2715−0.9843 NK5008pax 1 −1.4522 −1.6693 −1.3418 −1.5618 0.4085 −1.2843OL0003pax 1 −1.2897 −0.5083 −0.9542 −0.7241 −0.1385 −0.3214 OL0014pax 1−2.3397 −2.4483 −1.7342 −1.7191 −1.8585 −1.0564 OL0017pax 1 −1.0297−1.0083 −0.6692 −0.5541 0.6465 0.3036 OL0026pax 1 −2.2947 −1.4533−2.0092 −1.8291 −0.6635 −0.4364 OL0034pax 1 −1.3647 −0.8333 −1.2992−1.7341 0.1615 0.0536 OL0041pax 1 −2.2297 −1.6183 −2.1492 −2.8641−1.4835 −2.1464 OL0043pax 1 −0.8772 −0.6193 −0.7268 −1.2568 0.0285−0.5693 OL0052pax 1 −1.3047 −1.2733 −0.4242 −0.5541 −0.2135 −0.5464OL0056pax 1 −0.2972 −0.9943 −0.0518 −0.6268 1.5235 0.6557 OL0057pax 1−0.8447 −0.4883 −0.7942 −0.8191 −0.1335 −0.1664 OL0058pax 1 −1.1572−1.2643 −1.1518 −0.9918 −0.1265 −1.8993 OL0059pax 1 −1.1947 −0.0983−0.5492 0.0109 0.0115 0.0936 OL0060pax 1 −1.8622 −1.6843 −1.4368 −2.0668−1.6665 −1.0693 OL0062pax 1 0.3003 0.2817 0.3108 0.0709 1.7915 1.3986OL0063pax 1 −1.3172 −0.8843 −0.8068 −0.5168 −0.3665 −0.7093 OL0064pax 10.3203 0.9317 0.7908 0.6409 1.8015 0.7186 OL0065pax 1 0.4578 0.72570.6832 0.6982 1.8085 0.6357 OL0066pax 1 −0.2722 0.5757 −0.0618 0.42320.6785 1.0357 OL0068pax 1 −1.9622 −0.7143 −1.3218 −1.1918 −0.5765−1.0593 OL0070pax 1 −1.3622 −0.6193 −1.2868 −2.0718 −0.3665 −1.5243OL0071pax 1 −0.6897 0.1217 −0.3542 0.1559 0.7515 0.8136 OL0072pax 1−1.1047 −0.4033 −0.4392 −1.1791 0.2315 −0.9964 OL0073pax 1 −2.6897−1.9933 −1.1642 −1.1641 0.3565 −0.5114 OL0074pax 1 −1.4447 −0.7883−0.9142 −0.6891 1.0865 −0.1364 OL0075pax 1 −0.8322 −0.0343 −0.32180.3782 0.8585 0.5457 OL0077pax 1 −1.0747 −0.5933 0.1708 0.3059 0.22650.2036 OL0078pax 1 −2.2597 −1.8983 −1.1942 −1.1441 −0.8135 −0.9764OL0079pax 1 −1.1797 −0.7633 −0.9442 −1.2641 −0.8335 −0.0214 OL0080pax 1−0.0772 0.4507 0.2882 0.3882 0.3985 0.5357 PB3545pax 1 −0.3222 1.46570.0382 0.4682 1.1435 0.6807 PB3890pax 1 −0.4397 −0.2383 0.4108 0.77590.1265 0.1686

The test set results confirmed the surprising finding based on thetraining set that ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1 eachexpress RNA on average at a significantly higher level (p-value lessthan 0.05) in blood of subjects having colorectal cancer relative tosubjects having no colorectal pathology (Table 13). The ranges offold-change in the levels of RNA encoded by ANXA3, CLEC4D, LMNB1, PRRG4,TNFAIP6 and VNN1 normalized to levels of RNA encoded by IL2RB in bloodof the test set subjects having colorectal cancer relative to the testset subjects not having any colorectal pathology are also shown in Table13.

TABLE 13 Sample test set ranges of fold-changes in levels of RNA encodedby ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1 normalized to levels ofRNA encoded by IL2RB in blood of subjects having colorectal cancerrelative to subjects not having any colorectal pathology. Gene ANXA3CLEC4D LMNB1 PRRG4 TNFAIP6 VNN1 Average normalized RNA level in −0.630.01 −0.27 −0.38 0.56 0.28 subjects having colorectal cancer (ΔCt)Average normalized RNA level in 0.45 0.85 0.45 0.59 1.47 1.04 subjectsnot having any colorectal pathology (ΔCt) Average RNA level fold-change2.11 1.80 1.65 1.95 1.88 1.69 p-value for average RNA level fold-1.2E−17 7.3E−12 1.5E−15 2.5E−19 5.4E−12 2.6E−10 change Maximum observedRNA level 20.61 9.85 10.64 13.07 16.37 11.93 directional fold-change

As can be seen in Table 13, a test subject having a blood level of RNAencoded by ANXA3, normalized to a level of RNA encoded by IL2RB, whichis 2.1 to 20.6 fold higher than the average level of RNA encoded by thisgene in blood of subjects not having any colorectal pathology is morelikely to have colorectal cancer than to not have any colorectalpathology.

As can be seen in Table 13, a test subject having a blood level of RNAencoded by CLEC4D, normalized to a level of RNA encoded by IL2RB, whichis 1.8 to 9.85 fold higher than the average level of RNA encoded by thisgene in blood of subjects not having any colorectal pathology is morelikely to have colorectal cancer than to not have any colorectalpathology.

As can be seen in Table 13, a test subject having a blood level of RNAencoded by LMNB1, normalized to a level of RNA encoded by IL2RB, whichis 1.65 to 10.6 fold higher than the average level of RNA encoded bythis gene in blood of subjects not having any colorectal pathology ismore likely to have colorectal cancer than to not have any colorectalpathology.

As can be seen in Table 13, a test subject having a blood level of RNAencoded by PRRG4, normalized to a level of RNA encoded by IL2RB, whichis 1.95 to 13.1 fold higher than the average level of RNA encoded bythis gene in blood of subjects not having any colorectal pathology ismore likely to have colorectal cancer than to not have any colorectalpathology.

As can be seen in Table 13, a test subject having a blood level of RNAencoded by TNFAIP6, normalized to a level of RNA encoded by IL2RB, whichis 1.9 to 16.4 fold higher than the average level of RNA encoded by thisgene in blood of subjects not having any colorectal pathology is morelikely to have colorectal cancer than to not have any colorectalpathology.

As can be seen in Table 13, a test subject having a blood level of RNAencoded by VNN1, normalized to a level of RNA encoded by IL2RB, which is1.7 to 11.9 fold higher than the average level of RNA encoded by thisgene in blood of subjects not having any colorectal pathology is morelikely to have colorectal cancer than to not have any colorectalpathology.

Furthermore, the test set results confirmed the surprising finding basedon the training set that logistic regression models based on bloodexpression levels for any of the 63 possible combinations of one or moreof ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1, each of whichnormalized against expression levels of IL2RB, can be used todiscriminate, with a ROC AUC of at least 0.66 (Table 11), betweensubjects having colorectal cancer and subjects not having any colorectalpathology. As such, the novel logistic regression models listed in Table11 can be used to determine the probability that a test subject hascolorectal cancer as opposed to not having any colorectal pathology,based on blood levels of expression of ANXA3, CLEC4D, LMNB1, PRRG4,TNFAIP6 and/or VNN1 normalized to those of IL2RB.

Example 4 Determination of the Probability that a Test Subject hasColorectal Cancer as Opposed to not Having Colorectal Cancer Using BloodLevels of RNA Encoded by the Colorectal Cancer Markers: ANXA3, CLEC4D,IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1 Normalized to Those of ACTB

A blood sample from a test subject is analyzed for levels of RNA encodedby ACTB, ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1, asdescribed in Example 1, above, thereby generating test data. Logisticregression model #1 of Table 6 is applied to the test data, therebyproviding the probability that the test subject has colorectal cancer asopposed to not having any colorectal pathology.

Example 5 Determination of the Probability that a Test Subject hasColorectal Cancer as Opposed to not Having Colorectal Cancer Using BloodLevels of RNA Encoded by the Colorectal Cancer Markers: ANXA3, CLEC4D,IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1 Normalized to Those of IL2RB

A blood sample from a test subject is analyzed for levels of RNA encodedby ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1 as described inExample 1, above, thereby generating test data. Logistic regressionmodel #64 of Table 11 is applied to the test data, thereby providing theprobability that the test subject has colorectal cancer as opposed tonot having any colorectal pathology.

Example 6 Measurement of Blood Levels of RNA Encoded by a Combination ofANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1 Relative to the Level ofRNA Encoded by IL2RB can be Used to Determine the Probability that aTest Subject has Colorectal Cancer as Opposed to not Having anyColorectal Pathology

Materials and Methods:

Refer to “General materials and methods”, above.

Experimental Results:

Sample Training Set:

Discovery of Significantly Different Levels of RNA Encoded by ANXA3,CLEC4D, LMNB1, PRRG4, VNN1, TNFAIP6 Normalized to IL2RB in Blood ofSubjects Having Colorectal Cancer Relative to Subjects not Having anyColorectal Pathology:

Quantitative reverse transcriptase-PCR analysis of gene expression in atraining set of blood samples from 112 subjects having colorectal cancerand 120 subjects not having any colorectal pathology (subset of sampleslisted in Table 9 of Example 3, above), using IL2RB as duplex partnerfor normalization of gene expression levels was performed. Thenormalized RNA levels measured are shown in Table 14.

TABLE 14 Sample training set levels of RNA encoded by ANXA3, CLEC4D,IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1 in blood of subjects havingcolorectal cancer (Group 1) and subjects not having any colorectalpathology (Group 0), normalized to levels of RNA encoded by IL2RB.Levels shown correspond to ΔCt. Gene Sample ID Group ANXA3 CLEC4D LMNB1PRRG4 TNFAIP6 VNN1 CD0011pax 0 1.0600 1.5250 1.3250 1.1000 2.3500 2.7750CD0012pax 0 1.3600 1.6300 0.8600 0.8500 1.8350 2.7300 CD0030pax 0 1.41001.4500 0.9800 1.2850 1.0250 1.9600 CD0063pax 0 2.5700 3.3050 1.75501.9950 3.6000 3.2500 CD0077pax 0 −0.2350 0.7700 0.1500 0.3000 0.39001.1650 CD0078pax 0 1.5150 2.2800 0.7550 0.6750 2.6250 1.3750 CD0085pax 00.5750 1.6050 0.6450 0.9450 2.5450 0.7200 CD0117pax 0 0.9750 2.39000.9250 2.0600 2.4600 0.9400 CD0167pax 0 −0.9750 0.2000 0.4600 0.66001.6250 0.5800 CD0249pax 0 −0.5100 −0.2750 −0.5200 0.3200 0.2750 0.6400CD0286pax 0 −0.2850 1.0900 0.2500 0.2050 0.3550 0.3300 CD0297pax 00.0300 0.4850 0.4700 −0.0300 0.3900 1.0050 CD0323pax 0 1.9000 1.95000.8350 1.4850 2.6400 3.4450 CD0445pax 0 0.6600 0.8250 0.3000 0.37501.4100 1.4350 CD0463pax 0 −0.0850 1.7650 0.6000 0.8550 1.3700 1.5650CD0491pax 0 −0.5550 0.0650 0.1100 0.5450 0.9000 0.2150 CD0496pax 01.2050 2.8450 1.2400 1.9200 1.4650 1.3450 CD0501pax 0 1.1050 1.91001.1100 1.0600 2.8650 1.4900 CD0504pax 0 −0.7750 −0.2850 −0.1500 0.6100−0.1950 0.3350 CD0573pax 0 1.8100 1.2350 0.7700 0.8300 2.2650 1.8500CD0578pax 0 1.8200 1.9800 0.9550 1.0450 2.0700 2.2950 CD0639pax 0 0.29500.7900 0.7450 −0.2600 1.2000 1.7900 CD0645pax 0 −1.0950 −0.6000 −0.7700−0.6250 −0.0200 −0.1800 CD0679pax 0 0.2300 1.5950 0.5200 0.3350 1.88001.8750 CD0685pax 0 0.5250 1.7300 0.4350 0.9850 1.6850 1.0550 CD0716pax 01.9900 2.4700 0.8200 0.5250 1.4050 2.1600 CD0749pax 0 0.1600 1.06000.1200 0.3600 1.6800 0.8350 CD0760pax 0 −2.3750 −1.0500 −1.7400 −0.9100−0.8100 −0.6700 CD0811pax 0 1.9250 2.3300 0.7400 0.8900 0.9700 1.0100CD0848pax 0 1.0900 1.6300 1.4050 1.3850 1.9400 2.4750 CD0924pax 0 0.34500.7500 0.2250 −0.1100 0.5050 0.3050 CD1066pax 0 −0.2050 0.0950 −0.1200−0.1750 1.3650 0.1800 CD1073pax 0 0.3050 0.1350 0.3150 0.9350 1.08500.3400 CD1075pax 0 0.0300 0.9000 0.4100 1.5200 1.6000 0.8400 CD1089pax 0−1.3250 −0.2000 −1.1200 −0.8700 −0.1650 −0.2550 CD1116pax 0 −0.3850−0.3700 −0.4500 0.0001 1.0600 0.1750 CD1120pax 0 −1.1350 −0.0400 −0.74500.0150 1.0100 −0.5700 CD1198pax 0 0.2950 0.4300 0.6350 0.5200 0.78500.6000 PB1179pax 0 1.2100 1.2700 1.1550 1.2700 2.6850 1.7300 PB1277pax 00.6600 1.3300 0.2500 0.6650 1.6000 0.5900 PB1301pax 0 −2.1150 −1.4000−1.3850 −1.1000 −1.2350 −1.7950 PB1315pax 0 −1.3350 0.4800 −0.3200−0.1800 0.0600 1.0750 PB1345pax 0 0.0150 0.5500 0.5950 0.8350 0.36501.9650 PB1520pax 0 0.9250 2.0050 0.7850 −0.1800 1.4100 1.8550 PB1574pax0 1.2150 2.0550 1.2150 0.9500 1.3300 1.7150 PB1783pax 0 1.7400 1.84501.3600 1.3450 2.3150 1.8150 PB1799pax 0 0.7800 1.1900 0.6900 1.11502.3400 0.9800 PB1811pax 0 1.0950 1.6200 1.1050 1.4050 1.5800 1.2650PB1830pax 0 0.3450 0.8850 0.5650 0.2700 1.3300 2.4950 PB1833pax 0−0.0150 0.7050 0.0700 0.3150 0.5200 1.2600 PB1843pax 0 0.8750 0.94000.4750 0.8500 −0.3150 1.6500 PB1851pax 0 0.2450 −0.0450 0.1250 0.16502.6550 1.3150 PB1919pax 0 1.3100 1.8550 0.6050 1.5350 2.0800 2.2100PB1922pax 0 −0.1700 0.8350 −0.0700 0.8450 1.6350 0.5700 PB1924pax 00.0950 0.5700 −0.0750 0.2150 1.0050 1.3000 PB1937pax 0 1.5250 2.55001.3500 3.0050 2.7050 2.3350 PB1964pax 0 0.6950 2.7450 1.0200 1.67502.6250 3.0550 PB2027pax 0 0.0900 0.5100 0.3500 1.3200 1.5250 0.7550PB2029pax 0 0.5250 1.0200 0.6150 0.4300 2.2750 1.4350 PB2073pax 0 0.60001.0100 0.9000 1.8550 1.7200 1.2800 PB2099pax 0 −0.1500 −0.0950 0.13500.3250 0.0950 0.6700 PB2100pax 0 1.2350 1.3800 0.8950 1.5600 2.31001.4450 PB2132pax 0 0.5100 1.2400 0.1700 0.5100 1.9950 1.4750 PB2168pax 01.1400 1.4600 0.6600 1.0000 2.1650 1.2450 PB2192pax 0 −0.0050 0.4950−0.1450 −0.4800 0.8350 1.5800 PB2196pax 0 0.8850 1.7250 0.9850 1.90502.2550 1.1350 PB2200pax 0 0.8550 1.3850 0.2350 0.3050 0.9050 0.4400PB2213pax 0 1.8550 2.0000 0.9300 0.5300 2.9900 1.5850 PB2224pax 0−0.3850 0.4250 −0.1400 0.1000 1.8950 0.9750 PB2228pax 0 2.0300 2.22001.8000 2.0050 4.0300 3.0800 PB2229pax 0 0.0050 −0.0800 0.2000 −0.61500.9150 0.2550 PB2277pax 0 1.3400 1.0050 0.7550 0.3700 1.3100 1.7800PB2297pax 0 0.2900 0.8050 −0.0900 −0.0850 0.5250 −0.2800 PB2312pax 01.5250 2.1700 1.6550 1.2050 2.2900 2.1700 PB2398pax 0 −0.3800 0.22500.0150 −0.4700 0.1050 1.4250 PB2409pax 0 0.3950 1.0600 0.4950 0.50501.1550 1.7350 PB2414pax 0 3.0050 2.6050 1.4550 2.3150 3.9600 2.1150PB2467pax 0 0.4800 1.0800 0.3500 0.3600 2.1300 1.0850 PB2473pax 0 0.23500.8600 0.2850 0.8850 1.0100 1.5950 PB2512pax 0 0.8200 1.8950 1.18000.8200 2.0650 2.3750 PB2568pax 0 −0.2950 0.1900 −0.0900 0.5850 1.47501.1700 PB2571pax 0 0.5800 1.2800 0.7500 0.4300 2.0650 2.1050 PB2824pax 00.8400 1.3650 0.8000 1.3950 2.0150 1.7750 PB2880pax 0 1.2500 1.44000.8850 −0.0600 2.2550 1.8100 PB3088pax 0 1.3800 1.2900 0.8550 0.60502.9200 2.0800 RC0882pax 0 −0.0150 1.2350 0.2800 0.3700 1.7700 1.6550RC0888pax 0 −1.0450 −0.4250 −0.3400 0.0550 0.4950 0.1900 RC0968pax 0−0.6900 −0.4750 0.2700 1.5500 1.0250 2.3500 RC2114pax 0 −0.2000 0.96000.3300 0.6550 0.4550 2.0900 RC2238pax 0 1.3450 2.0600 0.8800 1.83002.7050 3.1100 RC2681pax 0 0.1100 0.6300 0.2200 0.2550 0.2550 0.6550RC2703pax 0 1.8000 2.1000 1.0500 1.5250 1.4250 1.5350 RC2749pax 0 0.09000.7750 −0.0850 0.0500 1.2550 1.2500 RC2750pax 0 −1.5150 −1.0750 −0.9000−0.2200 0.0700 −0.5400 RC2756pax 0 1.6800 1.3850 1.0550 1.4600 1.94502.5150 RC2771pax 0 −0.8450 0.4950 −0.6450 −0.4150 1.1200 1.2750RC2790pax 0 1.0850 1.1500 0.6150 0.8950 1.3350 1.5650 RC2792pax 0 1.01000.6250 0.6300 1.0950 1.9150 1.3550 RC2808pax 0 0.4850 1.4600 0.27000.5100 0.6550 1.0150 RC2822pax 0 0.4750 0.8350 −0.0900 0.3350 2.24000.4750 RC2834pax 0 −0.7350 0.2100 −0.6700 1.5700 2.1300 −0.3050RC2871pax 0 0.6150 1.1650 0.9250 1.3150 2.4200 2.2250 RC2879pax 0−0.4050 0.2050 −0.2400 0.5300 0.5250 0.0900 RC2892pax 0 −0.1500 0.1200−0.4050 0.2750 1.6700 0.9100 RC2895pax 0 1.8700 2.2100 1.3750 1.65001.5650 2.2500 RC2921pax 0 1.2850 1.4150 0.9900 1.4400 1.9950 2.0900RC2958pax 0 0.9250 1.0000 0.2300 0.0001 0.2700 1.2250 RC3022pax 0 0.1450−0.0150 0.1200 0.7850 1.3950 0.2450 RC3112pax 0 1.0250 1.2350 0.31500.2650 2.3150 0.8700 RC3146pax 0 −0.3350 −0.3450 −0.6200 0.8250 0.9650−0.3750 RC3184pax 0 2.4850 2.8750 1.3900 1.6250 2.5600 2.3950 RC3232pax0 −0.2550 0.7850 −0.2900 0.6900 −0.1250 2.6700 RC3324pax 0 0.3650 1.08000.1200 0.6100 1.5200 0.1250 RC3327pax 0 0.3600 0.0950 0.2250 −0.05001.4550 1.9700 RC3355pax 0 −0.1850 0.4200 −0.4950 −0.3100 1.2150 0.3400RC3380pax 0 −0.6450 0.3950 −0.5700 −0.3000 0.4950 −0.2300 RC3413pax 00.2750 0.8950 0.1250 −0.3400 0.5050 2.3050 RC3421pax 0 0.3550 0.46000.3900 0.4400 0.1300 0.2750 RC3468pax 0 −0.3600 −0.2800 −0.4100 −0.57500.9000 0.0350 RC3498pax 0 −0.3150 −0.7750 −0.7050 −0.2900 0.7750 −0.1800CC0003pax 1 −1.2200 −0.2350 −0.8100 −0.6450 1.0450 −0.6850 CD0157pax 11.2350 1.0300 0.7450 0.9100 2.5050 2.3600 CD0164pax 1 0.3150 0.85501.0100 1.2550 2.7950 1.8100 CD0256pax 1 −0.4000 0.2050 0.1950 1.04001.2350 0.3300 CD0322pax 1 −0.9950 −0.2450 −0.7150 −0.1400 0.6750 1.2850CD0356pax 1 −0.4950 −0.8850 −0.4500 −0.5650 −0.1000 −0.8600 CD0371pax 10.2350 1.0650 0.0500 −0.1800 0.9500 −0.0100 CD0629pax 1 0.1350 0.98500.7900 1.4800 0.5100 1.3850 CD1050pax 1 1.1750 0.3600 0.6850 1.02501.7150 1.3150 DS0003pax 1 −1.6350 −0.5200 −1.1950 −0.4900 −0.0650−0.0600 FC0005pax 1 −0.1950 −0.0950 0.0900 −0.0450 0.1950 1.4100FC0011pax 1 −0.0650 0.1250 0.1700 0.0800 1.1250 0.4950 FC0012pax 1−2.2350 −0.1250 −1.0650 −0.9850 −0.0150 −0.2000 JGA0001pax 1 −2.4550−1.7500 −1.8250 −0.8650 −1.4800 −1.4400 JH0002pax 1 0.3150 0.7350 0.28500.1350 1.1700 0.3000 JH0003pax 1 0.0550 0.0650 −0.3850 −0.6250 −0.14500.7100 JH0004pax 1 −0.0550 −0.0050 −0.0250 −0.2100 1.4050 1.0300JH0005pax 1 0.2950 1.3450 0.3700 0.4300 1.8650 1.2700 JH0006pax 1−0.4300 0.4450 −0.0050 −0.0850 1.2650 −0.0600 JH0007pax 1 −2.4350−1.9900 −1.3150 −0.1700 0.1250 −0.9150 JH0008pax 1 0.9050 2.5850 0.62501.1850 2.3350 1.0050 JH0009pax 1 −1.0700 −1.3450 −0.3350 −0.9650 −0.67000.1550 JH0010pax 1 −0.7650 0.0800 −0.1650 −0.0750 0.0300 −0.6650JH0012pax 1 −0.2150 0.1900 0.1000 −0.2150 0.4400 0.6950 JH0013pax 1−0.1200 0.3750 −0.0300 0.1150 2.2800 0.9200 JH0014pax 1 1.0050 0.89500.1500 −0.3400 1.6200 2.2850 JH0016pax 1 0.7850 1.1950 0.8600 0.43500.6450 1.0350 JH0018pax 1 −0.9850 −0.0250 −0.1450 −0.2900 0.0750 0.6250JH0019pax 1 0.2350 0.8450 0.3600 −0.0500 0.4700 0.4850 JH0020pax 10.0150 1.7550 0.3850 −0.3900 0.5100 1.0550 JH0021pax 1 −1.1000 0.3250−0.8650 −1.3850 −0.2350 −0.2400 JH0023pax 1 −0.4850 1.2250 0.3900 1.45001.0100 1.8450 JH0024pax 1 0.8350 2.0750 0.6950 1.0150 2.8900 2.4000JH0025pax 1 −0.9850 −0.5650 −0.6350 −1.2100 −0.3500 0.0950 JH0026pax 1−1.1000 0.7000 0.1500 0.1150 1.3050 1.0750 JH0027pax 1 −3.1650 −2.4400−2.2300 −1.9300 −2.2400 −1.4900 JH0028pax 1 0.5350 1.3350 1.2450 1.05502.8950 1.8900 JH0029pax 1 −1.5700 −0.9750 −0.9300 −1.1200 −0.9200 0.4200JH0031pax 1 −1.0400 −0.1700 −0.6950 −0.7700 0.0450 0.1250 JH0032pax 11.3700 1.6300 0.2650 0.1150 1.7300 1.7250 JH0033pax 1 −1.1900 0.5700−0.8700 −1.0850 0.9700 −0.5100 JH0034pax 1 −0.7000 0.8150 0.2850 0.13001.4900 1.2200 JH0035pax 1 0.0500 1.5700 0.4400 −0.2850 2.1400 1.1050JH0036pax 1 0.5650 0.9350 0.2450 0.0300 1.7450 0.8500 JH0038pax 1−0.0100 1.2650 −0.0100 0.0400 1.9650 0.4500 JH0039pax 1 0.2600 0.55500.3050 0.8450 3.0050 −0.1900 JH0040pax 1 0.3950 0.9000 0.3650 0.22501.1100 0.8850 JH0041pax 1 0.3800 1.2000 −0.0650 −0.3050 1.4900 0.6150JH0042pax 1 −2.4200 −0.9750 −1.4450 −0.8750 −1.4500 −1.6900 JH0043pax 1−0.4900 0.3050 0.0001 −0.6800 −0.0800 0.3250 JH0046pax 1 0.2350 0.59500.9350 0.0450 1.0300 0.5850 JH0047pax 1 0.3250 2.0950 1.0700 1.47001.8200 2.4300 JH0051pax 1 −0.6850 0.2250 −0.1950 −0.8800 −0.0700 −0.1750JH0052pax 1 −0.4500 0.5050 −0.1700 −0.6150 −0.1350 −0.2700 JH0053pax 1−1.2800 −0.5550 −0.9600 −0.3500 −0.6500 −0.1450 JH0057pax 1 −0.04502.1200 0.3050 0.7500 1.6350 1.4300 JH0059pax 1 −0.5200 0.4200 −0.0500−0.0650 0.6050 0.5050 JH0060pax 1 −0.7400 0.3250 0.0200 0.2900 0.66000.5100 JH0061pax 1 0.9900 2.8050 0.8700 1.4500 3.6400 0.8950 JH0063pax 1−0.6100 0.5650 −0.2300 −1.0050 0.2750 1.5400 JH0065pax 1 −2.8600 −1.5150−2.1500 −2.2300 −1.4500 −1.7100 JH0066pax 1 −1.3550 −0.3200 −1.1800−1.7900 −1.3500 −0.5400 JH0068pax 1 0.0050 0.4550 0.2550 0.0200 1.35001.8000 JH0069pax 1 −0.8650 0.2450 −0.2650 −0.5000 −0.0500 −0.6400JH0071pax 1 −2.5050 −2.2600 −1.8250 −0.3500 −1.3300 −1.5800 JH0072pax 10.1100 −0.5750 0.0350 −0.4650 1.3350 0.2300 JH0077pax 1 −0.1000 0.50000.4000 0.1150 0.8750 1.7850 JH0078pax 1 1.6350 1.6250 1.6300 0.66001.5100 1.7800 JH0080pax 1 −2.3200 −1.2350 −1.2600 −1.0250 −1.5600 0.0450JH0082pax 1 −0.9000 −0.6650 −0.3200 −0.5450 −0.3550 0.7200 JH0083pax 1−1.5800 −0.0750 −0.5300 −1.5900 0.2450 0.2050 JH0086pax 1 −0.2250−0.1850 −0.4800 −1.3250 −0.0850 0.6800 JH0092pax 1 −0.5450 1.3750 0.0400−0.1050 0.1300 1.1100 MH0001pax 1 1.4250 1.8900 1.5250 1.4350 3.13502.2700 MH0009pax 1 −0.2050 0.2150 −0.4850 −0.4450 −0.0400 −0.2050MH0012pax 1 0.0650 1.3100 0.5700 1.0950 1.4000 1.1200 MH0014pax 1 0.67001.1300 0.6100 0.2850 2.6850 1.5750 MH0016pax 1 −1.0950 −0.6050 −0.6750−1.2050 −0.2450 −0.4300 MH0017pax 1 −0.0100 0.8250 0.2000 −0.4150 1.25001.6250 MH0018pax 1 0.9650 0.6700 0.1850 0.2300 2.0650 0.5700 MH0021pax 10.9700 0.4800 −0.0650 0.2700 1.9100 1.8700 MH0022pax 1 0.2100 0.72500.1150 0.1250 0.8450 1.0950 MH0024pax 1 0.3450 0.6300 0.2050 −0.05500.3350 1.0900 MH0028pax 1 0.1350 0.5200 0.1350 −0.5950 0.1250 0.0850MH0029pax 1 0.2300 0.4850 0.5700 −0.2050 1.3250 0.9050 MH0035pax 10.8900 2.2000 1.2200 0.9950 1.3300 1.9450 MH0037pax 1 0.0001 1.34000.2600 0.3800 1.5200 2.0000 MH0038pax 1 1.5050 1.4150 1.1650 1.23002.1700 1.9850 MH0039pax 1 −1.4900 −0.7350 −0.5400 −0.8300 −0.4350 0.4500MH0042pax 1 −0.3500 −0.0800 0.0950 −0.2900 −0.1300 0.6500 MH0050pax 1−1.2600 0.7250 −0.4300 −0.2350 1.1300 1.8350 MH0051pax 1 −1.0300 −0.9000−0.5750 −1.4350 −1.9200 0.0300 MIP0004pax 1 −2.3200 −2.6150 −1.7200−1.5500 −1.3550 −2.5350 MP0013Apax 1 0.3150 1.0550 −0.2400 0.0200 1.2450−0.0700 MP0014Bpax 1 −0.2950 1.1200 −0.1400 0.2950 1.5600 0.3350MP0018Apax 1 −0.7700 −0.2050 −0.6850 −1.4250 −0.1050 −1.1150 MP0019Bpax1 −0.9100 −0.5150 −0.7550 −1.0250 −0.6750 0.7300 MP0024pax 1 −1.2900−0.1150 −0.4700 0.2100 1.3100 0.7400 NK2001pax 1 −0.6250 0.0850 −0.4250−1.2950 0.1750 1.6050 NK2002pax 1 −0.2400 −0.4300 −0.1450 −0.6550 0.29500.7000 NK2003pax 1 −0.4150 0.4150 0.0200 0.0950 1.4100 0.2550 NK2004pax1 −0.9100 0.3750 −0.6100 −0.8750 0.3500 −0.2700 PB1829pax 1 0.56001.7600 0.3700 0.2100 1.8100 1.9150 PB1842pax 1 1.2550 1.6150 1.15500.5300 2.5900 1.3450 PB1872pax 1 0.0650 0.3550 0.2400 −0.8650 0.86500.0950 PB2857pax 1 −0.8600 0.6800 −0.2350 0.3500 0.0450 1.2500 RC2919pax1 1.8500 1.8900 1.4100 2.7600 2.8200 3.8300 RC3062pax 1 −0.1750 0.1900−0.3350 −0.0750 −0.2800 −0.0100 RC3277pax 1 −0.4200 −0.1950 −0.5050−0.4350 0.3200 −0.0200 RC3297pax 1 0.0600 0.9800 0.1650 0.3850 2.56501.0450 RC3445pax 1 −1.0200 −0.4350 −0.6300 −0.9300 0.3100 0.1100RC3467pax 1 1.9450 2.9300 1.4200 1.2250 2.9400 2.6000

Surprisingly, analysis of the data showed that RNA encoded by ANXA3,CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1 is present on average at asignificantly higher level (p-value less than 0.05) in blood of subjectshaving colorectal cancer relative to subjects having no colorectalpathology (Table 15). The ranges of fold-change in the levels of RNAencoded by these genes normalized to levels of RNA encoded by IL2RB inblood of the training set subjects having colorectal cancer relative tothe training set subjects not having any colorectal pathology are shownin Table 15.

TABLE 15 Sample training set ranges of fold-change in levels of RNAencoded by ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1 normalized tolevels of RNA encoded by IL2RB in blood of subjects having colorectalcancer relative to subjects not having any colorectal pathology. GeneANXA3 CLEC4D LMNB1 PRRG4 TNFAIP6 VNN1 Average normalized RNA level in−0.32 0.41 0.78 −0.06 −0.13 0.64 subjects having colorectal cancer (ΔCt)Average normalized RNA level in 0.46 0.99 1.44 0.39 0.65 1.25 subjectsnot having any colorectal pathology (ΔCt) Average RNA level fold-change1.71 1.50 1.58 1.37 1.72 1.53 p-value for average RNA level fold-1.1E−08 1.0E−05 8.8E−06 2.3E−06 2.8E−12 6.3E−06 change Maximum observedRNA level 12.33 12.20 12.81 6.15 7.38 13.83 directional fold-change

As can be seen in Table 15, a test subject having a blood level of RNAencoded by ANXA3, normalized to a level of RNA encoded by IL2RB, whichis 1.7 to 12.3 fold higher than the average level of RNA encoded by thisgene in blood of subjects not having any colorectal pathology is morelikely to have colorectal cancer than to not have any colorectalpathology.

As can be seen in Table 15, a test subject having a blood level of RNAencoded by CLEC4D, normalized to a level of RNA encoded by IL2RB, whichis 1.5 to 12.2 fold higher than the average level of RNA encoded by thisgene in blood of subjects not having any colorectal pathology is morelikely to have colorectal cancer than to not have any colorectalpathology.

As can be seen in Table 15, a test subject having a blood level of RNAencoded by LMNB1, normalized to a level of RNA encoded by IL2RB, whichis 1.6 to 12.8 fold higher than the average level of RNA encoded by thisgene in blood of subjects not having any colorectal pathology is morelikely to have colorectal cancer than to not have any colorectalpathology.

As can be seen in Table 15, a test subject having a blood level of RNAencoded by PRRG4, normalized to a level of RNA encoded by IL2RB, whichis 1.4 to 6.2 fold higher than the average level of RNA encoded by thisgene in blood of subjects not having any colorectal pathology is morelikely to have colorectal cancer than to not have any colorectalpathology.

As can be seen in Table 15, a test subject having a blood level of RNAencoded by TNFAIP6, normalized to a level of RNA encoded by IL2RB, whichis 1.7 to 7.4 fold higher than the average level of RNA encoded by thisgene in blood of subjects not having any colorectal pathology is morelikely to have colorectal cancer than to not have any colorectalpathology.

As can be seen in Table 15, a test subject having a blood level of RNAencoded by VNN1, normalized to a level of RNA encoded by IL2RB, which is1.5 to 13.8 fold higher than the average level of RNA encoded by thisgene in blood of subjects not having any colorectal pathology is morelikely to have colorectal cancer than to not have any colorectalpathology.

Generation of a Logistic Regression Model (Optimized Relative to theModels Set Forth in Example 3 of the Examples Section, Above) forDetermining the Probability that a Test Subject has Colorectal CancerVersus not Having any Colorectal Pathology Via Measurement of Levels ofRNA Encoded by ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1 Normalizedto Levels of RNA Encoded by IL2RB:

Linear regression analysis of levels of RNA encoded by ANXA3, CLEC4D,LMNB1, PRRG4, TNFAIP6 and VNN1 normalized to IL2RB surprisingly showedthat a logistic regression model could be generated, based on bloodexpression levels normalized to IL2RB for the combination of these 6genes, for discriminating, with a ROC AUC of 0.80, between subjectshaving colorectal cancer and subjects not having any colorectalpathology (model #191 shown in Table 16).

The model of Table 16 corresponds to:

P={1+ê−[(0.126)+(−1.406)(L _(ANXA3))+(0.399)(L _(CLEC4D))+(1.874)(L_(LMNB1))+(−1.846)(L _(PRRG4))+(0.333)(L _(TNFAIP6))+(−0.277)(L_(VNN1))]}̂−1,

-   -   where P is the probability that a test subject has colorectal        cancer as opposed to not having any colorectal pathology, where        L_(ANXA3) is a ratio of a level of RNA encoded by ANXA3 to a        level of RNA encoded by IL2RB in blood of the test subject,        L_(CLEC4D) is a ratio of a level of RNA encoded by CLEC4D to a        level of RNA encoded by IL2RB in blood of the test subject,        L_(LMNB1) is a ratio of a level of RNA encoded by LMNB1 to a        level of RNA encoded by IL2RB in blood of the test subject,        L_(PRRG4) is a ratio of a level of RNA encoded by PRRG4 to a        level of RNA encoded by IL2RB in blood of the test subject,        L_(TNFAIP6) is a ratio of a level of RNA encoded by TNFAIP6 to a        level of RNA encoded by IL2RB in blood of the test subject, and        L_(VNN1) is a ratio of a level of RNA encoded by VNN1 to a level        of RNA encoded by IL2RB in blood of the test subject.

TABLE 16 Logistic regression model based on blood expression levels forthe combination of ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1,normalized to IL2RB expression levels for determining the probabilitythat a test subject has colorectal cancer as opposed to not havingcolorectal cancer. The ROC AUC value for the model is shown for thesample training set used to generate the models, as well as for anindependent blind sample test set used to test the model. No. ofGene-specific Logistic genes ROC AUC regression coefficient Regressionin Training Test Constant (K_(n)) Model # Model Set Set (K₀) ANXA3CLEC4D LMNB1 PRRG4 TNFAIP6 VNN1 191 6 0.80 0.80 0.126 −1.406 0.399 1.874−1.846 0.333 −0.277

Blind Sample Test Set:

Quantitative reverse transcriptase-PCR analysis of expression of ANXA3,CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1 in an independent test set ofblood samples from 202 subjects having colorectal cancer and 208subjects not having any colorectal pathology was performed as describedabove for the training set (these samples include a subset of thesamples listed in Table 12 of Example 3, above, as well as additionalsamples). The normalized RNA levels measured are shown in Table 17.

TABLE 17 Sample test set levels of RNA encoded by ANXA3, CLEC4D, LMNB1,PRRG4, TNFAIP6 and VNN1 in blood of subjects having colorectal cancer(Group 1) and subjects not having any colorectal pathology (Group 0),normalized to levels of RNA encoded by IL2RB. Levels shown correspond toΔCt. Gene Sample ID Group ANXA3 CLEC4D LMNB1 PRRG4 TNFAIP6 VNN1PB1952pax 0 2.240 2.125 0.965 2.285 3.220 2.520 RC3142pax 0 2.795 2.3801.225 2.040 2.675 2.500 CD1728pax 0 2.410 2.330 1.835 2.465 2.345 3.715PB2015pax 0 2.250 1.835 2.075 3.010 4.220 2.390 PB1786pax 0 1.210 2.5301.085 2.310 1.555 3.185 CD0762pax 0 1.215 1.260 1.140 2.360 1.925 1.730CD0800pax 0 2.410 2.310 1.550 2.210 3.480 2.795 PB3267pax 0 1.885 2.5651.285 2.180 2.325 2.575 PB2267pax 0 2.160 2.890 1.505 2.215 3.085 2.895CD0411pax 0 2.510 2.950 1.900 2.365 3.890 3.660 CD0211pax 0 1.115 2.2351.350 2.650 2.470 2.310 PB1918pax 0 2.450 2.080 1.145 1.590 3.675 2.435PB0701pax 0 1.220 2.090 1.760 2.655 2.190 3.785 PB3445pax 0 1.400 1.8701.015 1.955 2.280 2.205 PB1763pax 0 2.035 2.435 1.720 2.490 2.975 1.785PB3213pax 0 2.245 2.340 1.390 1.795 2.255 2.100 CD1424pax 0 1.460 1.3001.220 1.975 2.535 2.510 PB2978pax 0 2.945 3.175 1.990 2.175 3.680 2.840PB3270pax 0 1.895 3.055 0.885 1.415 1.280 2.545 RC2030pax 0 1.580 2.0601.205 1.595 1.665 4.080 CD0448pax 0 2.195 2.955 1.800 2.400 4.090 3.800RC2869pax 0 1.620 1.415 0.880 1.475 2.500 2.520 PB4296pax 0 1.940 2.6001.445 1.765 2.290 3.890 CD0398pax 0 2.425 2.560 1.970 2.265 4.015 3.515CD1077pax 0 0.740 0.845 0.575 1.560 1.410 1.560 PB3805pax 0 2.040 1.6551.515 1.605 1.020 1.610 PB1898pax 0 2.075 2.445 1.180 1.800 3.165 1.410PB2062pax 0 2.090 2.110 1.455 1.850 3.090 2.220 CD0937pax 0 1.120 0.7050.655 1.365 2.310 1.860 RC2612pax 0 2.725 2.685 1.810 1.830 3.395 2.535CD1784pax 0 1.615 1.410 1.325 1.735 2.325 2.325 PB2984pax 0 1.205 2.3250.990 1.895 2.270 2.315 RC2976pax 0 −0.045 1.445 −0.305 0.685 −0.3053.570 PB1785pax 0 1.470 1.950 1.245 1.880 1.835 1.450 CD0691pax 0 1.7852.850 1.445 2.060 2.535 1.900 PB2384pax 0 1.700 2.440 0.915 1.380 2.5002.405 CD1550pax 0 2.420 3.640 2.150 2.235 3.065 3.690 CD1540pax 0 1.0050.905 0.785 1.465 1.745 0.990 RC2565pax 0 −0.125 0.690 −0.330 0.7350.200 1.790 CD0499pax 0 1.390 2.750 0.805 1.720 2.820 1.290 RC2174pax 00.440 1.020 0.790 1.700 1.705 2.265 PB3304pax 0 0.550 1.270 0.520 1.7602.765 0.835 PB1879pax 0 1.345 1.065 1.295 1.620 2.070 1.830 PB3808pax 01.450 1.405 1.225 1.580 2.810 2.435 PB4357pax 0 1.065 0.195 0.295 0.6901.245 0.360 PB2636pax 0 1.795 2.615 1.360 1.775 3.600 2.925 PB3440pax 01.320 1.720 1.280 1.515 0.640 1.495 PB2272pax 0 2.225 2.130 1.335 1.4753.785 1.805 PB1848pax 0 1.410 1.150 0.765 0.875 2.300 2.775 RC3420pax 01.265 1.490 1.390 2.060 2.875 0.995 PB2005pax 0 1.800 1.830 1.045 1.2853.465 2.250 CD0354pax 0 0.680 1.160 0.585 1.395 2.015 1.265 CD1945pax 01.005 1.500 0.260 0.815 2.430 2.055 PB4156pax 0 2.005 2.405 1.435 1.3701.995 1.990 RC2934pax 0 1.055 1.185 0.605 1.025 2.760 2.560 PB2214pax 01.350 1.790 0.535 0.980 2.390 1.315 PB3370pax 0 0.965 2.430 1.125 1.7951.985 2.235 PB1300-2pax 0 0.885 1.535 0.425 0.960 1.765 1.860 PB2951pax0 −0.180 −1.980 −1.215 −0.650 −0.520 −0.915 PB3356pax 0 −0.430 1.050−0.145 1.105 0.680 1.550 CD0148pax 0 0.740 1.890 0.515 1.255 1.835 1.665PB3451pax 0 1.240 1.300 0.720 1.025 2.210 1.555 CD1409pax 0 0.510 0.1150.140 0.495 0.660 1.250 PB3118pax 0 1.480 1.615 1.415 1.375 1.195 1.855PB3931pax 0 1.460 1.515 0.925 1.250 2.790 1.210 CD1163pax 0 1.105 2.1101.475 2.175 3.175 1.875 RC2112pax 0 1.255 2.285 1.555 1.830 1.825 2.530PB4274pax 0 0.670 0.385 0.075 0.245 0.535 1.735 CD1028pax 0 1.250 2.1150.985 1.370 2.240 1.950 PB4345pax 0 0.680 1.445 0.895 1.320 1.125 2.145CD0698pax 0 0.925 1.555 0.915 1.295 1.265 1.515 PB4066pax 0 0.855 0.1750.580 0.940 1.450 0.185 PB4062pax 0 0.720 1.380 0.455 1.305 2.385 0.300PB3481pax 0 −0.110 0.835 0.185 1.025 0.445 1.345 CD0252pax 0 1.100 0.3400.380 0.615 2.185 0.580 CD0428pax 0 0.480 1.090 0.225 0.815 1.420 1.465CD0571pax 0 1.060 1.425 1.430 1.960 2.575 0.890 CD0786pax 0 0.850 1.3350.795 1.290 2.045 1.325 PB3863pax 0 1.745 1.785 1.435 1.255 2.620 2.605PB2927pax 0 1.955 0.820 0.520 −0.140 1.515 1.890 PB3568pax 0 1.505 1.6251.250 1.295 2.155 1.370 RC2839pax 0 0.690 1.275 0.595 0.935 1.195 1.800CD1700pax 0 1.095 1.520 1.160 1.250 1.480 2.020 CD1313pax 0 1.600 1.4000.710 0.820 2.670 0.510 PB1700pax 0 0.690 1.020 0.740 1.155 1.610 1.265CD0727pax 0 1.515 2.780 1.015 1.245 2.130 1.450 PB4161pax 0 0.940 0.9450.755 0.890 1.340 1.330 CD1583pax 0 0.690 0.855 0.530 0.780 1.240 1.445PB3594pax 0 0.885 1.175 0.500 0.935 2.395 1.040 RC3170pax 0 0.860 1.6350.190 0.210 0.400 2.110 CD0553pax 0 2.075 3.365 1.750 1.505 2.725 3.125CD0220pax 0 1.080 2.120 1.110 1.210 1.380 2.345 CD0238pax 0 0.355 1.0100.440 1.065 1.720 1.180 CD0409pax 0 −0.235 0.710 −0.100 0.650 0.5051.320 PB3163pax 0 0.530 1.630 0.885 1.515 2.675 2.245 PB2491pax 0 0.835−0.035 0.295 0.605 2.505 0.150 PB4377pax 0 0.110 0.315 0.030 0.565 1.2451.270 PB4307pax 0 1.845 3.790 1.730 1.775 3.650 3.665 RC2236pax 0 0.9701.080 1.035 0.985 1.450 2.095 RC2716pax 0 0.490 0.730 0.845 1.360 2.4901.365 PB2024pax 0 0.815 1.375 0.785 1.075 2.445 1.850 CD0484pax 0 0.1401.135 0.255 1.080 1.585 0.255 RC2897pax 0 0.385 1.035 0.130 0.530 1.3501.335 CD0872pax 0 0.580 −0.005 0.490 0.585 1.435 1.005 PB1626pax 0 0.5250.835 0.435 0.550 1.075 1.865 CD1974pax 0 0.965 1.445 0.350 0.555 2.0000.985 CD1295pax 0 0.560 0.450 0.425 0.560 1.575 1.570 RC2699pax 0 0.8251.015 0.340 0.395 1.385 1.285 RC2986pax 0 1.095 1.315 0.540 0.625 2.6751.710 PB1899pax 0 −0.230 0.420 −0.365 0.095 0.270 1.625 PB3955pax 01.630 1.745 1.220 1.070 3.000 1.545 PB1230pax 0 0.685 1.585 0.855 0.9550.950 1.870 CD1404pax 0 −0.045 1.190 0.225 0.740 1.330 2.630 CD0367pax 01.050 0.940 0.745 0.705 2.105 1.360 PB3226pax 0 0.720 1.225 0.760 1.0102.320 1.735 PB3193pax 0 0.475 0.650 0.650 0.905 1.600 1.205 PB3224pax 00.400 −0.115 0.080 0.125 0.415 0.390 PB1871pax 0 0.715 0.640 0.565 0.5451.590 1.710 CD1392pax 0 1.055 1.540 0.860 1.030 2.995 1.720 CD0833pax 0−0.050 −0.365 −0.090 0.365 0.630 −0.285 CD0386pax 0 1.070 2.390 1.0001.280 3.245 2.350 CD1158pax 0 0.335 0.270 0.110 0.065 0.185 1.405PB1324pax 0 −0.110 0.585 0.465 1.050 1.765 1.840 CD1455pax 0 −0.6100.530 0.345 1.150 0.755 1.570 RC2338pax 0 1.395 2.355 1.590 1.460 2.5152.445 CD1971pax 0 0.645 1.425 0.060 0.110 1.400 2.170 CD1048pax 0 0.8602.110 1.115 1.525 2.600 1.210 CD0244pax 0 0.285 0.305 0.260 0.670 1.395−0.095 RC3191pax 0 1.560 2.415 1.510 1.210 2.285 2.395 PB3582pax 0 1.0901.830 1.230 1.320 2.870 1.955 CD0237pax 0 0.595 1.085 0.245 0.400 1.4000.985 CD1981pax 0 1.955 3.135 1.875 1.495 3.520 3.250 CD0603pax 0 1.0151.015 0.640 0.400 1.755 1.770 CD1134pax 0 0.795 1.655 0.815 0.790 1.8502.475 PB2130pax 0 0.665 0.890 0.335 0.260 0.895 1.155 PB1275pax 0 −0.8850.195 −0.445 0.145 −0.230 2.010 PB2564pax 0 0.385 1.960 0.460 0.8101.055 1.265 CD0580pax 0 1.150 1.580 0.470 0.400 2.330 1.165 PB0768pax 0−0.670 0.740 0.080 0.825 0.425 1.335 CD0518pax 0 1.575 3.770 1.750 1.7753.120 2.440 RC3315pax 0 0.640 1.565 0.650 1.035 2.680 0.940 CD1270pax 00.895 1.020 0.770 0.515 1.695 1.680 CD1068pax 0 0.380 0.200 0.700 0.9852.475 0.400 CD0995pax 0 0.805 1.465 0.560 0.615 2.395 1.435 CD1438pax 0−0.125 0.620 −0.030 0.440 0.905 0.045 CD1169pax 0 0.270 0.295 0.4700.500 1.845 1.430 RC3379pax 0 0.850 1.185 0.460 0.410 1.945 0.400CD0520pax 0 −0.125 1.025 0.990 1.325 1.130 1.370 PB1718pax 0 0.145 1.1850.395 0.445 0.695 1.505 PB2757pax 0 0.555 1.690 0.995 0.915 1.750 2.335CD0743pax 0 0.290 −0.400 0.095 0.070 1.515 −0.120 CD0667pax 0 −0.385−0.305 −0.345 −0.065 0.205 −0.230 RC3214pax 0 0.940 1.860 1.200 1.1402.730 1.620 PB0689pax 0 0.235 1.420 0.295 0.270 0.905 1.990 CD0911pax 0−0.550 −0.580 −0.800 −0.405 0.290 −0.560 PB2516pax 0 0.310 1.075 0.7000.490 0.890 2.285 PB2584pax 0 0.945 1.230 0.935 0.475 1.270 1.340CD1487pax 0 0.310 2.120 1.190 1.645 2.160 0.815 CD0282pax 0 0.165 0.6850.270 0.545 1.100 −0.700 PB4325pax 0 0.065 −0.180 −0.020 −0.025 0.475−0.360 RC2652pax 0 −1.425 0.025 −0.620 0.450 0.760 0.560 PB2464pax 00.620 1.330 0.510 0.045 1.230 2.810 PB3227pax 0 0.235 0.850 0.820 0.8652.215 1.845 CD1559pax 0 0.170 1.000 0.335 0.505 2.070 1.300 PB4003pax 00.070 0.655 0.170 0.215 0.880 0.690 CD0108pax 0 0.260 0.055 0.560 0.3100.740 0.640 PB1758pax 0 −0.600 0.475 0.030 0.535 0.500 0.145 CD0277pax 00.605 1.980 0.800 0.940 2.250 0.720 PB2184pax 0 −1.005 −0.650 −0.705−0.445 −0.075 1.230 CD1683pax 0 0.485 1.620 0.725 0.575 1.595 1.805RC2615pax 0 −0.655 0.070 0.210 0.340 0.190 1.205 CD1224pax 0 −0.2950.630 −0.325 −0.110 0.995 0.485 CD1458pax 0 0.035 −0.055 0.560 0.4851.635 0.585 CD0204pax 0 0.380 0.885 0.250 −0.050 1.200 0.990 CD1706pax 00.540 1.815 1.025 1.020 2.510 1.195 CD1542pax 0 0.375 0.870 0.900 0.6551.755 1.305 PB2909pax 0 −0.345 −1.240 −0.675 −0.915 −0.075 −0.645PB4073pax 0 −0.250 0.195 −0.095 −0.205 0.410 0.600 CD0604pax 0 0.4151.130 0.320 0.235 2.125 0.230 PB3605pax 0 −1.120 −0.290 −0.695 −0.3400.305 0.660 CD1965pax 0 0.030 1.310 0.400 0.140 1.155 2.275 CD0432pax 00.185 0.565 0.015 −0.140 1.870 0.495 PB1336pax 0 0.695 0.780 0.320−0.470 1.310 1.805 PB2974pax 0 −0.635 0.320 −0.300 −0.305 0.605 1.740PB0662pax 0 0.120 0.575 0.945 0.850 1.605 0.045 CD1741pax 0 −0.310−0.335 0.015 −0.160 1.205 0.850 PB2875pax 0 −0.230 1.205 0.140 0.1651.200 1.025 CD0419pax 0 0.440 1.220 0.810 0.655 2.375 −0.030 CD1649pax 00.925 1.565 1.000 0.130 1.550 1.630 CD1329pax 0 0.130 −0.170 0.155−0.460 1.075 0.705 CD0466pax 0 0.730 1.870 0.930 0.160 1.280 1.890CD0857pax 0 −1.460 −0.755 −0.560 −0.360 −0.670 0.255 CD0242pax 0 0.9651.360 0.580 −0.450 0.980 1.235 PB3513pax 0 −0.355 −0.705 −0.970 −1.2000.410 −1.270 CD0583pax 0 −0.605 −1.045 −0.595 −0.740 0.765 −0.705PB3049pax 0 −1.030 0.045 −0.235 −0.170 0.105 0.595 PB1446pax 0 −1.380−0.485 −0.715 −0.495 0.300 0.700 CD1441pax 0 −0.565 0.485 0.230 0.1750.995 0.055 PB2634pax 0 0.165 1.335 1.270 1.000 2.740 1.020 CD0547pax 0−1.740 −0.260 −0.685 −0.185 −0.095 −0.420 PB2041pax 0 −0.655 0.440−0.205 −0.170 1.540 0.185 PB1514pax 0 −0.940 0.075 −0.280 −0.265 0.8500.175 PB3032pax 0 −0.145 0.435 0.750 0.110 1.510 1.425 CD0676pax 0−0.730 −0.640 −0.610 −1.085 0.300 0.100 PB3806pax 0 −1.410 0.325 −0.305−0.080 −0.010 −0.070 CD0472pax 0 −1.800 −0.180 −0.830 −0.465 0.255−0.070 PB1222pax 0 −0.165 0.415 0.325 −0.795 −0.035 0.860 CD1032pax 0−2.740 −2.045 −0.550 −0.490 −0.875 0.410 JH0111pax 1 1.040 1.910 0.8352.170 2.645 1.650 MH0122Bpax 1 0.235 1.190 0.340 1.380 0.430 3.590IS3001pax 1 1.925 2.145 1.030 1.300 1.885 2.880 BE3003pax 1 0.985 1.1601.050 1.985 2.185 1.120 MH0083pax 1 1.570 1.635 0.805 1.195 2.150 1.765BE1004pax 1 0.085 1.050 0.730 1.825 2.040 1.790 MH0112Bpax 1 0.960 1.7200.810 1.325 1.975 2.055 BE1007pax 1 1.050 0.945 0.815 1.055 1.810 2.100MH0031-2pax 1 −0.325 −0.130 −0.430 0.615 1.330 1.170 MH0112Apax 1 0.9251.760 1.025 1.500 2.025 2.220 MR4001Apax 1 0.610 1.130 0.470 1.210 1.6500.615 NK2011pax 1 1.355 2.025 1.060 1.110 2.520 3.570 OL0092pax 1 0.7901.555 0.670 1.160 1.585 1.650 MIP2007pax 1 1.075 1.725 0.855 1.265 2.4101.980 JH0153pax 1 1.085 1.470 0.610 0.580 0.495 2.085 KW0005pax 1 0.6951.540 1.240 1.940 2.705 1.850 JH0054pax 1 0.555 1.775 0.870 1.580 1.9901.710 AN4017pax 1 1.190 1.630 0.590 0.510 0.620 1.665 MP0031Apax 1 1.0901.315 0.705 0.800 1.720 1.570 MP0021Bpax 1 0.410 0.995 0.710 1.220 1.7651.555 MIP1018Bpax 1 0.600 1.535 0.490 1.020 1.720 1.115 CC0005pax 10.655 0.940 0.345 0.725 1.460 0.345 NK1009pax 1 1.915 1.845 1.030 0.6502.595 1.635 JM0010pax 1 0.130 0.610 0.670 1.075 1.260 1.775 JH0150pax 10.895 1.930 0.490 0.685 0.900 0.730 IS1004pax 1 1.495 2.710 1.750 1.5202.570 3.730 MIP1021Bpax 1 0.600 2.605 0.860 1.455 2.305 2.180 NK5006pax1 0.895 1.205 0.645 0.620 2.095 2.495 MP0032Bpax 1 0.630 1.060 0.3150.640 1.770 0.865 BE3012Apax 1 −0.830 −0.545 −1.045 −0.330 −0.330 0.700JH0146pax 1 −0.130 0.615 0.175 0.805 1.540 1.665 NK2007pax 1 0.110 0.520−0.030 0.560 0.980 −0.125 JH0112pax 1 0.500 0.910 0.265 0.530 1.6351.090 OL0066pax 1 0.140 1.050 0.380 0.785 1.140 1.410 DC0012pax 1 −1.370−0.520 −0.775 0.275 −1.140 −0.350 JH0129pax 1 0.580 1.530 0.935 1.2652.850 2.270 JH0163pax 1 0.295 1.490 0.955 1.580 2.625 1.310 FS0005pax 10.040 0.315 0.500 0.890 1.355 0.850 DES1006Apax 1 0.955 1.140 0.8900.635 1.570 1.940 MH0030pax 1 −0.150 0.865 0.180 0.720 0.345 0.260FS0006pax 1 0.100 0.195 0.585 0.825 1.165 1.025 OL0096pax 1 0.305 1.2300.890 0.830 0.405 2.550 MIP2003pax 1 −0.045 0.665 0.295 0.835 1.8651.060 MH0113Apax 1 0.495 0.085 0.800 0.575 1.015 1.565 JH0067pax 1 0.2300.940 0.100 0.355 1.180 0.980 MH0053pax 1 2.195 2.465 1.420 0.670 3.3152.550 MW0001Apax 1 0.345 1.160 0.540 0.680 1.290 1.545 AN4013pax 1 1.1352.150 1.055 0.770 1.810 2.225 WJ0005CSpax 1 −0.895 −0.630 −1.440 −0.8400.055 0.250 JH0096pax 1 −1.745 −0.480 −1.130 −0.400 −2.050 1.375DC5008Bpax 1 0.045 0.855 0.140 0.300 1.415 2.280 MIP2002pax 1 1.1201.680 1.060 0.755 2.000 1.890 AN0011pax 1 1.110 1.075 0.125 −0.780 0.3202.935 DES1009Apax 1 0.305 0.660 0.325 0.440 0.955 0.370 DC0011pax 10.150 0.700 0.615 0.730 1.535 1.880 FC0013pax 1 −0.105 −0.095 0.9200.965 1.255 2.145 OL0075pax 1 −0.590 0.190 −0.010 0.590 1.140 0.970NK2014pax 1 0.485 1.240 0.650 0.745 1.705 1.100 KW0008pax 1 0.090 0.9850.305 0.250 0.220 1.730 OL0080pax 1 −0.445 −0.025 −0.150 0.110 0.0300.540 AN4019pax 1 1.520 2.265 1.045 0.765 3.090 1.225 JH0116pax 1 0.0351.465 0.520 0.830 1.040 1.120 JH0132pax 1 −0.460 0.010 −0.310 0.0950.365 −0.005 JH0170pax 1 2.050 2.960 1.645 0.855 3.655 3.370 NK1001pax 10.415 0.990 −0.050 −0.025 1.805 0.975 OL0065pax 1 0.280 0.730 0.4450.530 1.630 0.690 BE3011Apax 1 0.640 1.825 0.600 0.680 2.150 1.105KW0004pax 1 0.895 2.485 1.415 1.290 1.975 1.935 JH0022pax 1 −0.265 0.2550.040 0.145 0.375 0.945 JH0100pax 1 0.550 1.420 1.020 0.955 1.955 1.405CD1690pax 1 0.120 0.145 0.110 0.165 1.040 −0.315 MH0063pax 1 0.265−0.295 −0.305 −0.215 2.185 −0.590 MIP1017pax 1 −0.260 0.825 −0.250 0.0000.670 0.800 JH0109pax 1 1.190 2.160 1.000 0.405 2.055 2.640 CC0001pax 10.170 0.765 0.295 0.315 1.785 1.305 PB3545pax 1 −0.520 1.150 −0.3800.170 0.795 0.475 IS4001pax 1 −0.855 −0.065 −0.915 −0.770 −0.370 1.630DC0005pax 1 1.085 1.960 1.220 0.715 1.895 1.965 MH0070pax 1 0.380 0.9450.685 0.515 1.575 1.395 JH0114pax 1 −1.875 −1.485 −1.400 −0.540 −1.710−1.970 CD1351pax 1 0.510 0.515 0.015 −0.295 1.830 0.785 OL0093pax 1−2.055 −0.120 −1.075 0.025 −0.430 0.560 JH0058pax 1 −0.610 0.305 0.1650.240 −0.060 1.195 DC5008Apax 1 −0.395 0.160 −0.190 −0.155 1.135 1.540OL0085pax 1 −0.985 −0.050 −0.750 −0.290 0.505 0.520 FS1022pax 1 −0.5300.295 0.400 0.675 0.655 0.040 OL0063pax 1 −0.915 −0.430 −0.335 −0.0250.055 −0.085 MH0108Apax 1 0.215 0.810 0.225 −0.005 0.885 0.570MIP2006pax 1 −0.590 0.470 −0.025 0.265 0.505 0.080 NK2016pax 1 −0.2200.425 0.075 0.215 1.975 0.835 OL0057pax 1 −0.975 −0.970 −1.340 −1.210−0.630 −0.575 JH0137pax 1 0.265 1.395 0.095 −0.125 1.190 1.175 JH0118pax1 0.030 −0.565 0.010 −0.405 0.610 0.125 MH0097pax 1 −0.785 −0.180 −0.195−0.015 0.345 0.430 JH0147pax 1 0.770 2.080 0.915 0.855 3.475 1.255BE4002pax 1 0.160 0.420 0.015 −0.445 0.760 1.265 MH0036pax 1 −0.0900.620 0.370 0.240 1.430 1.440 JH0142pax 1 −0.585 0.220 −0.185 0.0550.285 −0.605 MIP2010Apax 1 0.285 0.555 −0.075 −0.280 1.550 −0.070JH0169pax 1 −0.305 0.255 −0.120 −0.320 0.455 1.110 FS1010pax 1 0.4351.705 1.210 0.800 1.520 2.145 JGA0036pax 1 −0.515 0.390 −0.595 −0.995−0.725 2.090 MH0079pax 1 0.395 0.625 0.275 −0.375 0.500 1.020 OL0064pax1 0.030 0.500 0.305 0.120 1.345 0.580 JH0135pax 1 −1.480 −1.890 −1.505−1.320 −1.655 −1.480 OL0017pax 1 −0.790 −0.650 −0.375 −0.315 0.900 0.765CC2001pax 1 −0.285 1.450 0.335 0.480 1.315 1.210 DC6002Apax 1 −0.5700.975 0.200 0.395 0.930 1.000 JH0149pax 1 0.485 1.165 0.240 −0.065 2.3950.980 DC2009pax 1 −0.715 0.050 0.195 0.155 −0.350 0.365 MP0033Bpax 10.145 1.045 0.320 0.075 1.285 0.715 MP0030Bpax 1 −0.875 0.445 0.2700.690 2.285 1.790 JH0133pax 1 −0.335 0.500 0.045 −0.220 0.210 1.150OL0059pax 1 −1.335 −0.180 −0.735 −0.290 −0.065 −0.145 AN4026Bpax 1−0.175 0.485 0.215 −0.160 0.510 1.130 AN4022Apax 1 −0.540 0.830 −0.290−0.355 0.020 0.710 OL0077pax 1 −1.055 −0.525 −0.085 0.035 0.200 0.165OL0094pax 1 −0.305 0.535 0.190 0.015 0.710 0.460 KW0002pax 1 −0.6250.165 −0.505 −0.695 0.470 1.215 MH0066pax 1 −1.180 −0.855 −0.710 −0.595−0.320 −0.480 DC5006Apax 1 −0.210 0.160 0.105 −0.385 −0.220 0.280NK4001pax 1 −0.975 1.135 −0.090 0.260 0.500 0.810 JGA0029pax 1 −0.8700.590 −0.575 −0.195 1.295 0.185 MW0008Apax 1 −1.080 −0.505 −0.480 −0.440−0.300 −0.285 JH0131pax 1 −0.640 −0.890 −0.935 −1.555 −1.150 0.180JH0106pax 1 −0.720 −0.200 −0.105 −0.290 0.845 1.115 OL0056pax 1 −0.495−1.310 −0.470 −0.925 1.175 0.450 MH0076pax 1 0.515 1.955 0.540 −0.0752.340 2.570 NK2005pax 1 −1.575 −1.090 −0.925 −1.055 −2.160 −0.020MP0029Apax 1 −1.700 −0.870 −1.100 −0.670 0.275 0.060 MH0080pax 1 −0.3100.215 0.340 0.120 1.530 0.510 FS1014pax 1 −0.580 0.050 −0.030 0.0451.850 −0.105 JH0110pax 1 −0.565 0.185 −0.375 −0.830 −0.420 0.735JH0145pax 1 −2.030 −0.365 −0.940 −0.500 −1.170 0.445 MH0125Apax 1 −0.5501.265 0.135 −0.170 0.465 2.310 OL0058pax 1 −0.935 −1.050 −0.840 −0.8700.115 −1.495 DC0008pax 1 0.545 0.990 0.665 −0.070 1.875 0.815 MH0073pax1 −1.290 −0.595 −1.165 −1.040 0.040 −0.285 MIP0002pax 1 −2.285 −0.925−1.240 −0.595 −0.575 0.055 JH0138pax 1 −0.680 0.150 −0.065 −0.315 0.1200.340 OL0074pax 1 −1.255 −0.530 −0.680 −0.410 1.280 0.145 OL0079pax 1−0.920 −0.125 −0.510 −0.755 −0.360 0.460 MR1001pax 1 −0.120 0.795 0.015−0.490 0.650 0.495 DES5001Bpax 1 −1.335 0.395 −1.095 −0.735 0.770 0.315MH0093pax 1 −2.215 −1.375 −1.240 −0.880 −0.800 0.255 MP0027Apax 1 −0.4150.765 −0.140 −0.350 0.940 0.200 IS1005Apax 1 −1.180 −1.030 −0.340 −0.4450.325 −0.025 KW0003pax 1 −0.295 0.830 0.335 −0.580 −0.325 2.740CD1260pax 1 −0.730 −1.005 −0.325 −0.795 0.725 0.510 JH0130pax 1 −1.110−0.400 −0.940 −1.210 −0.505 0.220 MH0096pax 1 −1.225 0.790 −0.670 −0.4200.865 0.665 OL0052pax 1 −1.075 −1.005 −0.060 −0.315 0.040 −0.265MIP0005pax 1 −2.020 −1.140 −1.305 −0.835 −0.420 −1.190 OL0014pax 1−2.000 −2.250 −1.480 −1.640 −1.645 −0.705 WJ0003RTpax 1 −1.935 −1.860−1.760 −1.735 −1.070 −1.055 MIP2013Bpax 1 0.025 1.825 0.110 −0.890−0.500 1.975 JH0164pax 1 −0.115 0.335 0.015 −0.615 1.275 0.005 JH0123pax1 −2.050 −1.365 −1.125 −0.920 −1.165 −1.315 OL0089pax 1 −1.415 0.130−0.455 −0.365 −0.305 −0.520 DES1013Bpax 1 −1.130 0.105 −0.450 −0.5950.160 0.065 AN4011pax 1 −0.155 0.805 0.770 0.280 2.110 0.880 JH0093pax 10.095 1.090 0.085 −0.695 1.290 0.885 OL0043pax 1 −0.705 −0.475 −0.555−1.105 0.230 −0.335 JH0097pax 1 −0.280 −0.285 0.270 −0.445 0.780 −0.415DC3003pax 1 −0.265 0.975 0.030 −0.625 1.600 1.845 OL0068pax 1 −1.790−0.720 −1.340 −1.230 −0.665 −0.895 KW0006pax 1 −1.320 0.575 0.155 0.0800.700 1.540 NK5008pax 1 −1.150 −1.435 −1.000 −1.300 0.610 −1.040DC6001Apax 1 −0.965 0.725 −0.225 −0.670 0.135 1.500 OL0034pax 1 −1.155−0.515 −0.985 −1.375 0.485 0.545 MH0062pax 1 −1.040 −0.245 −0.205 −0.6000.450 0.300 MIP1019Apax 1 −1.415 −0.495 −1.060 −1.125 0.800 −0.060MR1002pax 1 −0.890 0.295 0.315 −0.070 0.645 0.355 MH0090pax 1 −0.9950.240 −0.365 −0.705 0.000 −0.495 NK2010pax 1 −1.505 −1.070 −0.985 −1.320−0.870 −1.045 NK2009pax 1 0.060 0.745 0.470 −0.370 1.920 0.300 NK2018pax1 −2.160 −1.415 −1.155 −1.090 −0.640 −1.100 JH0144pax 1 0.475 2.5601.160 0.215 2.295 1.575 OL0078pax 1 −2.540 −2.250 −1.570 −1.555 −1.350−1.325 MIP1009pax 1 −2.950 −1.590 −2.030 −1.500 −0.685 −1.165 OL0025pax1 −2.010 −0.060 −0.940 −0.855 −0.380 −0.445 MH0078pax 1 −0.910 0.290−0.405 −1.410 −0.335 1.345 KW0007pax 1 −0.655 0.930 0.090 −0.640 0.8300.495 AN0013pax 1 0.060 1.905 0.875 −0.085 2.740 2.020 JH0076pax 1−0.535 −0.795 −0.360 −1.735 −0.300 −0.065 OL0090pax 1 −1.105 0.150 0.090−0.745 0.075 0.990 JH0120pax 1 −0.040 0.595 0.215 −0.935 1.860 0.390JH0126pax 1 −1.570 −0.110 −0.455 −1.020 −0.435 0.415 JH0113pax 1 −0.0550.805 0.605 −0.715 0.835 0.570 JH0085pax 1 −0.960 −0.445 −0.075 −1.0950.185 0.295 MH0081pax 1 0.390 1.375 0.640 −0.845 2.085 1.300 AN0001pax 1−1.830 −1.010 −0.840 −1.320 0.110 −0.175 MH0095pax 1 −2.145 −1.475−1.350 −1.795 −0.915 −0.830 DS0007pax 1 −3.365 −2.210 −1.965 −1.820−3.555 −2.965 JH0136pax 1 −2.355 −1.670 −0.970 −1.215 −0.845 −1.710OL0072pax 1 −1.135 −0.315 −0.375 −1.170 0.415 −0.875 BE1006pax 1 −2.635−1.570 −1.580 −1.735 −0.580 −0.740 JH0048pax 1 −2.285 −0.250 −1.315−1.350 −1.075 −2.025 OL0073pax 1 −2.980 −2.425 −1.650 −1.685 −0.100−0.650 FS0002pax 1 −2.275 −1.035 −0.730 −1.030 −0.780 −1.700 OL0041pax 1−1.890 −1.030 −1.595 −2.325 −0.970 −1.465 AN0007pax 1 −3.105 −1.775−2.300 −3.180 −2.690 −1.255

The test set results confirmed the surprising finding based on thetraining set that ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1 eachexpress RNA on average at a significantly higher level (p-value lessthan 0.05) in blood of subjects having colorectal cancer relative tosubjects having no colorectal pathology (Table 18). The ranges offold-change in the levels of RNA encoded by ANXA3, CLEC4D, LMNB1, PRRG4,TNFAIP6 and VNN1 normalized to levels of RNA encoded by IL2RB in bloodof the test set subjects having colorectal cancer relative to the testset subjects not having any colorectal pathology are also shown in Table18.

TABLE 18 Sample test set ranges of fold-changes in levels of RNA encodedby ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1 normalized to levels ofRNA encoded by IL2RB in blood of subjects having colorectal cancerrelative to subjects not having any colorectal pathology. Gene ANXA3CLEC4D LMNB1 PRRG4 TNFAIP6 VNN1 Average normalized RNA level in subjects−0.37 0.39 −0.04 −0.07 0.76 0.74 having colorectal cancer (ΔCt) Averagenormalized RNA level in subjects 0.72 1.21 0.65 0.90 1.73 1.51 nothaving any colorectal pathology (ΔCt) Average RNA level fold-change 2.121.77 1.61 1.97 1.95 1.70 p-value for average RNA level fold-change2.8E−24 3.0E−14 3.3E−19 1.9E−26 5.0E−17 2.4E−12 Maximum observed RNAlevel directional 16.95 12.44 7.71 16.96 38.96 22.19 fold-change

As can be seen in Table 18, a test subject having a blood level of RNAencoded by ANXA3, normalized to a level of RNA encoded by IL2RB, whichis 2.1 to 17.0 fold higher than the average level of RNA encoded by thisgene in blood of subjects not having any colorectal pathology is morelikely to have colorectal cancer than to not have any colorectalpathology.

As can be seen in Table 18, a test subject having a blood level of RNAencoded by CLEC4D, normalized to a level of RNA encoded by IL2RB, whichis 1.8 to 12.4 fold higher than the average level of RNA encoded by thisgene in blood of subjects not having any colorectal pathology is morelikely to have colorectal cancer than to not have any colorectalpathology.

As can be seen in Table 18, a test subject having a blood level of RNAencoded by LMNB1, normalized to a level of RNA encoded by IL2RB, whichis 1.6 to 7.7 fold higher than the average level of RNA encoded by thisgene in blood of subjects not having any colorectal pathology is morelikely to have colorectal cancer than to not have any colorectalpathology.

As can be seen in Table 18, a test subject having a blood level of RNAencoded by PRRG4, normalized to a level of RNA encoded by IL2RB, whichis 2.0 to 17.0 fold higher than the average level of RNA encoded by thisgene in blood of subjects not having any colorectal pathology is morelikely to have colorectal cancer than to not have any colorectalpathology.

As can be seen in Table 18, a test subject having a blood level of RNAencoded by TNFAIP6, normalized to a level of RNA encoded by IL2RB, whichis 2.0 to 39.0 fold higher than the average level of RNA encoded by thisgene in blood of subjects not having any colorectal pathology is morelikely to have colorectal cancer than to not have any colorectalpathology.

As can be seen in Table 18, a test subject having a blood level of RNAencoded by VNN1, normalized to a level of RNA encoded by IL2RB, which is1.7 to 22.2 fold higher than the average level of RNA encoded by thisgene in blood of subjects not having any colorectal pathology is morelikely to have colorectal cancer than to not have any colorectalpathology.

Furthermore, the test set results confirmed the surprising finding basedon the training set that logistic regression model #191 based on bloodexpression levels of the combination of ANXA3, CLEC4D, LMNB1, PRRG4,TNFAIP6 and VNN1, each of which normalized against expression levels ofIL2RB, can be used to discriminate, with a ROC AUC of at least 0.80(Table 16), between subjects having colorectal cancer and subjects nothaving any colorectal pathology. As such, the novel logistic regressionmodel #191 can be used to determine the probability that a test subjecthas colorectal cancer as opposed to not having any colorectal pathology,based on blood levels of expression of ANXA3, CLEC4D, LMNB1, PRRG4,TNFAIP6 and/or VNN1 normalized to those of IL2RB.

Example 7 Determination of the Probability that a Test Subject hasColorectal Cancer as Opposed to not Having Colorectal Cancer Using BloodLevels of RNA Encoded by the Colorectal Cancer Markers: ANXA3, CLEC4D,IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1 Normalized to Those of IL2RB

A blood sample from a test subject is analyzed for levels of RNA encodedby ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1 as described inExample 1, above, thereby generating test data. Logistic regressionmodel #191 of Table 16 is applied to the test data, thereby providingthe probability that the test subject has colorectal cancer as opposedto not having any colorectal pathology.

All patents, patent applications, and published references cited hereinare hereby incorporated by reference in their entirety.

One skilled in the art will appreciate readily that the invention iswell adapted to carry out the objects and obtain the ends and advantagesmentioned, as well as those objects, ends and advantages inherentherein. The present examples, along with the methods, procedures,treatments, molecules, and specific compounds described herein arepresently representative of preferred embodiments, are exemplary, andare not intended as limitations on the scope of the invention. Changestherein and other uses will occur to those skilled in the art which areencompassed within the spirit of the invention as defined by the scopeof the claims.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is:
 1. A kit comprising packaging and containing, foreach gene of a set of two or more genes selected from the groupconsisting of ACTB, ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 andVNN1, a primer set capable of generating an amplification product of DNAcomplementary to RNA encoded, in a human subject, only by the gene. 2.The kit of claim 1, further containing two or more components selectedfrom the group consisting of a thermostable polymerase, a reversetranscriptase, deoxynucleotide triphosphates, nucleotide triphosphatesand enzyme buffer.
 3. The kit of claim 1, further containing at leastone labeled probe capable of selectively hybridizing to either a senseor an antisense strand of the amplification product.
 4. The kit of claim1, further containing a computer-readable medium having instructionsstored thereon that are operable when executed by a computer forapplying a mathematical model to test data representing a level of RNAencoded by the gene in blood of a human test subject, wherein themathematical model is derived from positive control data representinglevels of RNA encoded by the gene in blood of human control subjectshaving colorectal cancer, and from negative control data representinglevels of RNA encoded by the gene in blood of human control subjects nothaving colorectal cancer, wherein the mathematical model is fordetermining a probability that data representing a level of RNA encodedby the gene corresponds to the positive control data and not to thenegative control data, and wherein the probability that the test datacorresponds to the positive control data and not to the negative controldata represents the probability that the test subject has colorectalcancer as opposed to not having colorectal cancer.
 5. The kit of claim1, further containing a computer-readable medium having instructionsstored thereon that are operable when executed by a computer forapplying, to test data representing a level of RNA encoded by the genein blood of a human test subject and to negative control datarepresenting a level of RNA encoded by the gene in blood of humancontrol subjects not having colorectal cancer, a mathematical formulafor generating a value indicating, for ANXA3, CLEC4D, LMNB1, PRRG4,TNFAIP6 and VNN1, whether the level of RNA encoded by the gene in bloodof the test subject is higher than the level of RNA encoded by the genein blood of human control subjects not having colorectal cancer, and,for IL2RB, whether the level of RNA encoded by the gene in blood of thetest subject is lower than the level of RNA encoded by the gene in bloodof human control subjects not having colorectal cancer, wherein, forANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6 and VNN1, an indication by thevalue that the level of RNA encoded by the gene in blood of the testsubject is higher than the level of RNA encoded by the gene in blood ofhuman control subjects not having colorectal cancer classifies the testsubject as more likely to have colorectal cancer than to not havecolorectal cancer, and wherein, for IL2RB, an indication by the valuethat the level of RNA encoded by the gene in blood of the test subjectis lower than the level of RNA encoded by the gene in blood of humancontrol subjects not having colorectal cancer classifies the testsubject as more likely to have colorectal cancer than to not havecolorectal cancer.
 6. The primer set of claim 1, wherein the set ofgenes consists of IL2RB and PRRG4.
 7. The primer set of claim 1, whereinthe set of genes consists of ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6and VNN1.
 8. A method of determining a probability that a human testsubject has colorectal cancer as opposed to not having colorectalcancer, the method comprising, for each gene of a set of one or moregenes selected from the group consisting of ANXA3, CLEC4D, IL2RB, LMNB1,PRRG4, TNFAIP6 and VNN1: (a) determining a level of RNA encoded by thegene in blood of the test subject, thereby generating test data; (b)providing positive control data representing levels of RNA encoded bythe gene in blood of human control subjects having colorectal cancer,and providing negative control data representing levels of RNA encodedby the gene in blood of human control subjects not having colorectalcancer; and (c) determining a probability that the test data correspondsto the positive control data and not to the negative control data,wherein the probability that the test data corresponds to the positivecontrol data and not to the negative control data represents theprobability that the test subject has colorectal cancer as opposed tonot having colorectal cancer.
 9. The method of claim 8, furthercomprising determining levels of RNA encoded by the gene in blood of apopulation of human subjects having colorectal cancer, thereby providingthe positive control data representing the levels of RNA encoded by thegene in blood of human control subjects having colorectal cancer, anddetermining levels of RNA encoded by the gene in blood of a populationof human subjects not having colorectal cancer, thereby providing thenegative control data representing the levels of RNA encoded by the genein blood of human control subjects not having colorectal cancer.
 10. Themethod of claim 8, wherein the determining of the probability that thetest data corresponds to the positive control data and not to thenegative control data is effected by applying to the test data amathematical model derived from the positive control data and from thenegative control data, and wherein the mathematical model is fordetermining the probability that data representing a level of RNAencoded by the gene corresponds to the positive control data and not tothe negative control data.
 11. A method of classifying a human testsubject as more likely to have colorectal cancer than to not havecolorectal cancer, the method comprising, for each gene of a set of oneor more genes selected from the group consisting of ANXA3, CLEC4D,IL2RB, LMNB1, PRRG4, TNFAIP6 and VNN1: (a) determining a level of RNAencoded by a CLEC4D gene in blood of the test subject, therebygenerating test data; (b) providing negative control data representing alevel of RNA encoded by the gene in blood of human control subjects nothaving colorectal cancer; and (c) applying to the test data and to thenegative control data a mathematical formula for generating a valueindicating whether the level of RNA encoded by the gene in blood of thetest subject is higher than the level of RNA encoded by the gene inblood of human control subjects not having colorectal cancer, wherein anindication by the value that the level of RNA encoded by the gene inblood of the test subject is higher than the level of RNA encoded by thegene in blood of human control subjects not having colorectal cancerclassifies the test subject as more likely to have colorectal cancerthan to not have colorectal cancer.
 12. The method of claim 11, furthercomprising determining levels of RNA encoded by the gene in blood of apopulation of human subjects not having colorectal cancer, therebyproviding the negative control data representing the levels of RNAencoded by the gene in blood of human control subjects not havingcolorectal cancer.
 13. The method of claim 8, wherein the set of genesconsists of IL2RB and PRRG4.
 14. The method of claim 8, wherein the setof genes consists of ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 andVNN1.
 15. The method of claim 8, wherein the determining of the level ofRNA encoded by the gene in blood of the test subject is effected bydetermining the level of RNA encoded by the gene in a blood sampleisolated from the test subject.
 16. The method of claim 8, wherein thelevel of RNA encoded by the gene in blood of the test subject isdetermined as a ratio to a level of RNA encoded by IL2RB in blood of thetest subject.
 17. The method of claim 11, wherein the set of genesconsists of IL2RB and PRRG4.
 18. The method of claim 11, wherein the setof genes consists of ANXA3, CLEC4D, IL2RB, LMNB1, PRRG4, TNFAIP6 andVNN1.
 19. The method of claim 11, wherein the determining of the levelof RNA encoded by the gene in blood of the test subject is effected bydetermining the level of RNA encoded by the gene in a blood sampleisolated from the test subject.
 20. The method of claim 11, wherein thelevel of RNA encoded by the gene in blood of the test subject isdetermined as a ratio to a level of RNA encoded by IL2RB in blood of thetest subject.